HPV16 E7 Oncoprotein Research Articles

A preliminary study on the immune responses of HPV16-E7 by combined intranasal immunization with lymphotoxin

Human papillomavirus (HPV) ranks the first cause of cervical cancer. Cervical cancer has high prevalence rates in women around the world. The HPV-E7 oncoprotein is expressed in cervical cancer and is a target of developing immunotherapies against HPV-associated tumors. However, the antigenicity of this protein is low. Due to this reason, potent adjuvants are required to enhance its therapeutic efficacy. This preliminary study aims to evaluate whether lymphotoxin (LT) could act as an effective immune adjuvant for HPV infection in mice models. Intranasal immunization was used to explore the effect of HPV-E7 and/or LT immune response. After the third intranasal immunization, the titer for the HPV-E7 antibody was detected in serum and vaginal washing fluid. Also, we assessed the expression of chemokine ligand 13 (CXCL13) and Peripheral Node Addressin (PNAd) in the lymph nodes after intranasal immunization with immunohistochemical analysis. compared to HPV-E7 immunization, intranasal immunization with HPV-E7 plus LT significantly increased HPV-E7-specific serum IgG and vaginal IgA titers. Furthermore, the combined use of HPV-E7 and LT strongly induced E7-specific CTL responses. LT can be effective for intranasal immunized HPV-E7 to improve E7-specific immune responses to HPV infection. It is new approach to eradicate chronic HPV infection capable of inducing an effective anti-infection method.

Liposomes as tunable platform to decipher the antitumor immune response triggered by TLR and NLR agonists

Liposomes are powerful tools for the optimization of peptides and adjuvant composition in cancer vaccines. Here, we take advantage of a liposomal platform versatility to develop three vaccine candidates associating a peptide from HA influenza virus protein as CD4 epitope, a peptide from HPV16 E7 oncoprotein as CD8 epitope and TLR4, TLR2/6 or NOD1 agonists as adjuvant. Liposomal vaccine containing MPLA (TLR4 liposomes), are the most effective treatment against the HPV-transformed orthotopic lung tumor mouse model, TC-1. This vaccine induces a potent Th1-oriented antitumor immunity, which leads to a significant reduction in tumor growth and a prolonged survival of mice, even when injected after tumor appearance. This efficacy is dependent on CD8+ T cells. Subcutaneous injection of this treatment induces the migration of skin DCs to draining lymph nodes. Interestingly, TLR2/6 liposomes trigger a weaker Th1-immune response which is not sufficient for the induction of a prolonged antitumor activity. Although NOD1 liposome treatment results in the control of early tumor growth, it does not extend mice survival. Surprisingly, the antitumor activity of NOD1 vaccine is not associated with a specific adaptive immune response. This study shows that our modulable platform can be used for the strategical development of vaccines.

Development of multiepitope therapeutic vaccines against the most prevalent high-risk human papillomaviruses.

Aim: Our goal was the development of DNA- or peptide-based multiepitope vaccines targeting HPV E7, E6 and E5 oncoproteins in tumor mouse model. Materials &methods: After designing the multiepitope E7, E6 and E5 constructs from four types of high risk HPVs (16, 18, 31 & 45) using bioinformatics tools, mice vaccination was performed by different homologous and heterologous modalities in a prophylactic setting. Then, anti-tumor effects of the best prophylactic strategies were studied in a therapeutic setting. Results: In both prophylactic and therapeutic experiments, groups receiving homologous E7+E6+E5 polypeptide, and heterologous E7+E6+E5 DNA prime/polypeptide boost were successful in complete rejection of tumors. Conclusion: The designed multiepitope constructs can be considered as promising candidates to develop effective therapeutic HPV vaccines.

Dual Specificity PDZ- and 14-3-3-Binding Motifs: A Structural and Interactomics Study

Protein-protein interaction motifs are often alterable by post-translational modifications. For example, 19% of predicted human PDZ domain-binding motifs (PBMs) have been experimentally proven to be phosphorylated, and up to 82% are theoretically phosphorylatable. Phosphorylation of PBMs may drastically rewire their interactomes, by altering their affinities for PDZ domains and 14-3-3 proteins. The effect of phosphorylation is often analyzed by performing "phosphomimetic" mutations. Here, we focused on the PBMs of HPV16-E6 viral oncoprotein and human RSK1 kinase. We measured the binding affinities of native, phosphorylated, and phosphomimetic variants of both PBMs toward the 266 human PDZ domains. We co-crystallized all the motif variants with a selected PDZ domain to characterize the structural consequence of the different modifications. Finally, we elucidated the structural basis of PBM capture by 14-3-3 proteins. This study provides novel atomic and interactomic insights into phosphorylatable dual specificity motifs and the differential effects of phosphorylation and phosphomimetic approaches.

Human Papilloma Virus Increases ΔNp63α Expression in Head and Neck Squamous Cell Carcinoma.

P63, and in particular the most expressed ΔNp63α isoform, seems to have a critical role in the outcome of head and neck cancer. Many studies have been conducted to assess the possible use of p63 as a prognostic marker in squamous cell carcinoma cancers, but the results are still not well-defined. Moreover, a clear relationship between the expression of ΔNp63α and the presence of high-risk HPV E6 and E7 oncoproteins has been delineated. Here we describe how ΔNp63α is mostly expressed in HPV-positive compared to HPV-negative head and neck cancer cell lines, with a very good correlation between ΔNp63α mRNA and protein levels.

HPV16 E7-impaired keratinocyte differentiation leads to tumorigenesis via cell cycle/pRb/involucrin/spectrin/adducin cascade.

Here, we used codon usage technology to generate two codon-modified human papillomavirus (HPV)16 E7 genes and, together with wild-type E7, to construct three HPV16 E7 gene plasmids: Wt-E7, HB1-E7, and HB2-E7. The three HPV 16 E7 plasmids were used to investigate how HPV16 E7 protein was expressed in different cells and how this oncoprotein deregulated cellular and molecular events in human keratinocytes to induce carcinogenesis. We discovered that codon usage of HPV16 E7 gene played a key role in determining expression of E7 oncoprotein in all tested cells. HPV16 E7 inhibited significantly expression of pRb to impair keratinocyte differentiation and disrupted development of skin epidermis in mice. HPV16 E7 increased substantially the number of G0/G1 cells associated with upregulation of cyclin D2 and downregulation of cyclin B1 in keratinocytes. HPV16 E7 not only inhibited expression of involucrin and α-spectrin but also disrupted the organization of involucrin filaments and spectrin cytoskeleton. Furthermore, HPV16 E7 inhibited expression of β-adducin, destroyed its cytoskeletal structure and induced phosphorylation of β-adducin(Ser662) in keratinocytes. Importantly, HPV16 E7 induced carcinogenesis in mice associated with expression of phosphorylated β-adducin(Ser662) and its nucleus-translocation. In conclusion, we provided evidence that HPV16 E7 oncoprotein inhibited keratinocyte differentiation in vitro and in vivo leading to carcinogenesis through cell cycle arrest and disruption of pRb/involucrin/spectrin/adducin cascade.

Human papillomavirus oncoproteins and post-translational modifications: generating multifunctional hubs for overriding cellular homeostasis.

Human papillomaviruses (HPVs) are major human carcinogens, causing around 5% of all human cancers, with cervical cancer being the most important. These tumors are all driven by the two HPV oncoproteins E6 and E7. Whilst their mechanisms of action are becoming increasingly clear through their abilities to target essential cellular tumor suppressor and growth control pathways, the roles that post-translational modifications (PTMs) of E6 and E7 play in the regulation of these activities remain unclear. Here, we discuss the direct consequences of some of the most common PTMs of E6 and E7, and how this impacts upon the multi-functionality of these viral proteins, and thereby contribute to the viral life cycle and to the induction of malignancy. Furthermore, it is becoming increasingly clear that these modifications, may, in some cases, offer novel routes for therapeutic intervention in HPV-induced disease.

Human papillomavirus type 18 E5 oncoprotein cooperates with E6 and E7 in promoting cell viability and invasion and in modulating the cellular redox state.

BACKGROUND High-risk human papillomaviruses (HR-HPVs) are the etiological agents of cervical cancer. Among them, types 16 and 18 are the most prevalent worldwide. The HPV genome encodes three oncoproteins (E5, E6, and E7) that possess a high transformation potential in culture cells when transduced simultaneously. In the present study, we analysed how these oncoproteins cooperate to boost key cancer cell features such as uncontrolled cell proliferation, invasion potential, and cellular redox state imbalance. Oxidative stress is known to contribute to the carcinogenic process, as reactive oxygen species (ROS) constitute a potentially harmful by-product of many cellular reactions, and an efficient clearance mechanism is therefore required. Cells infected with HR-HPVs can adapt to oxidative stress conditions by upregulating the formation of endogenous antioxidants such as catalase, glutathione (GSH), and peroxiredoxin (PRX).OBJECTIVES The primary aim of this work was to study how these oncoproteins cooperate to promote the development of certain cancer cell features such as uncontrolled cell proliferation, invasion potential, and oxidative stress that are known to aid in the carcinogenic process.METHODS To perform this study, we generated three different HaCaT cell lines using retroviral transduction that stably expressed combinations of HPV-18 oncogenes that included HaCaT E5-18, HaCaT E6/E7-18, and HaCaT E5/E6/E7-18.FINDINGS Our results revealed a statistically significant increment in cell viability as measured by MTT assay, cell proliferation, and invasion assays in the cell line containing the three viral oncogenes. Additionally, we observed that cells expressing HPV-18 E5/E6/E7 exhibited a decrease in catalase activity and a significant augmentation of GSH and PRX1 levels relative to those of E5, E6/E7, and HaCaT cells.MAIN CONCLUSIONS This study demonstrates for the first time that HPV-18 E5, E6, and E7 oncoproteins can cooperate to enhance malignant transformation.

PDZ Domain-Containing Protein NHERF-2 Is a Novel Target of Human Papillomavirus 16 (HPV-16) and HPV-18.

Cancer-causing human papillomavirus (HPV) E6 oncoproteins have a class I PDZ-binding motif (PBM) on their C termini, which play critical roles that are related to the HPV life cycle and HPV-induced malignancies. E6 oncoproteins use these PBMs to interact with, to target for proteasome-mediated degradation, a plethora of cellular substrates that contain PDZ domains and that are involved in the regulation of various cellular pathways. In this study, we show that both HPV-16 and HPV-18 E6 oncoproteins can interact with Na+/H+ exchange regulatory factor 2 (NHERF-2), a PDZ domain-containing protein, which among other cellular functions also behaves as a tumor suppressor regulating endothelial proliferation. The interaction between the E6 oncoproteins and NHERF-2 is PBM dependent and results in proteasome-mediated degradation of NHERF-2. We further confirmed this effect in cells derived from HPV-16- and HPV-18-positive cervical tumors, where we show that NHERF-2 protein turnover is increased in the presence of E6. Finally, our data indicate that E6-mediated NHERF-2 degradation results in p27 downregulation and cyclin D1 upregulation, leading to accelerated cellular proliferation. To our knowledge, this is the first report to demonstrate that E6 oncoproteins can stimulate cell proliferation by indirectly regulating p27 through targeting a PDZ domain-containing protein.IMPORTANCE This study links HPV-16 and HPV-18 E6 oncoproteins to the modulation of cellular proliferation. The PDZ domain-containing protein NHERF-2 is a tumor suppressor that has been shown to regulate endothelial proliferation; here, we demonstrate that NHERF-2 is targeted by HPV E6 for proteasome-mediated degradation. Interestingly, this indirectly affects p27, cyclin D1, and CDK4 protein levels and, consequently, affects cell proliferation. Hence, this study provides information that will improve our understanding of the molecular basis for HPV E6 function, and it also highlights the importance of the PDZ domain-containing protein NHERF-2 and its tumor-suppressive role in regulating cell proliferation.

Antibodies against HPV16E6 oncoprotein in the Swiss HIV cohort study: Kinetics and anal cancer risk prediction.

Our aim was to describe HPV16E6 antibody kinetics prior to anal cancer in people living with HIV/AIDS (PLWHA) and evaluate the possible contribution of HPV16E6 serology to anal cancer risk prediction. For 91 persons diagnosed with anal cancer in the Swiss HIV Cohort Study (1989-2017), serial serum/plasma samples were tested for HPV16E6 antibodies using multiplex serology, supplemented with samples from 1,356 participants without anal cancer. Anal cancer incidence was estimated for PLWHA from 40 years-old in the cART era, stratified by HPV16E6 serostatus. HPV16E6 seroprevalence was 23.3% in samples <2 years prior to anal cancer diagnosis and decreased with increasing time prior to cancer: 16.7% at 2-4 years, 4.4% at 5-9, and 7.0% at ≥10 years. Of 25 individuals with anal cancer who were HPV16E6-seropositive at any time during follow-up, the majority (n = 18) remained seropositive in all samples after seroconversion, whereas for seven cases, seropositivity was transitory. Among individuals with anal cancer, HPV16E6 seroprevalence was marginally higher in women vs. men who have sex with men (adjusted OR = 4.3, 95% CI: 1.1, 17.2) and in older participants (adjusted OR = 6.2, 95% CI: 1.1, 34.8 for cases diagnosed at ≥55 vs. <45 years). Anal cancer incidence was 402/100,000 person-years in HPV16E6-positive vs. 82/100,000 in HPV16E6-negative PLWHA (incidence rate ratio = 4.9, 95% CI: 1.3, 13.1). In conclusion, HPV16E6 serology, despite its low sensitivity, allows characterization of a group of individuals with very high anal cancer incidence and may have a place in secondary prevention in groups at high risk for anal cancer such as PLWHA.

Human papillomavirus 16 promotes microhomology-mediated end-joining

Squamous cell carcinomas (SCCs) arising from aerodigestive or anogenital epithelium that are associated with the human papillomavirus (HPV) are far more readily cured with radiation therapy than HPV-negative SCCs. The mechanism behind this increased radiosensitivity has been proposed to be secondary to defects in DNA repair, although the specific repair pathways that are disrupted have not been elucidated. To gain insight into this important biomarker of radiosensitivity, we first examined genomic patterns reflective of defects in DNA double-strand break repair, comparing HPV-associated and HPV-negative head and neck cancers (HNSCC). Compared to HPV-negative HNSCC genomes, HPV+ cases demonstrated a marked increase in the proportion of deletions with flanking microhomology, a signature associated with a backup, error-prone double-strand break repair pathway known as microhomology-mediated end-joining (MMEJ). Then, using 3 different methodologies to comprehensively profile double-strand break repair pathways in isogenic paired cell lines, we demonstrate that the HPV16 E7 oncoprotein suppresses canonical nonhomologous end-joining (NHEJ) and promotes error-prone MMEJ, providing a mechanistic rationale for the clinical radiosensitivity of these cancers.

Induction of co-inhibitory molecule CTLA-4 by human papillomavirus E7 protein through downregulation of histone methyltransferase JHDM1B expression

Human papillomavirus causes various skin diseases and even cancer. Unfortunately, the host immune system often fails to generate effective responses against HPV infection due to the ability of HPV to evade immune-mediated eradication, although the detailed mechanisms by which HPV inhibits host antiviral immunity are not fully understood. In this study, we reported a novel role of HPV E7 oncoprotein in inducing the expression of co-inhibitory molecule cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) in cells of epithelial origin. Mechanistically, HPV E7 protein downregulated the cellular abundance of Jumonji C histone demethylase 1B (JHDM1B), increasing the levels of H3K36 methylation within the promoter region of CTLA-4. Our findings expand the current understanding of HPV-mediated immune evasion mechanisms and may be helpful in developing optimal anti-HPV therapeutic strategies and relevant drugs.

A genetic variant within MDM4 3′UTR miRNA binding site is associated with HPV16‐positive tumors and survival of oropharyngeal cancer

As mouse double minute 4 (MDM4) and HPV16 E6 oncoproteins play important roles in inhibition of p53 activity, a functional polymorphism (rs4245739) in the 3' untranslated regions of MDM4 targeted by microRNA-191 may alter its expression level or functional efficiency, thus affecting tumor status and survival in human papillomavirus (HPV)-positive squamous cell carcinoma of oropharynx (SCCOP). A total of 564 incident SCCOP patients with definitive radiotherapy were included for determination of tumor HPV16 status and genotypes of the polymorphism. Univariate and multivariable Cox models were performed to assess the associations between the polymorphism and outcomes. We found that MDM4 rs4245739 had statistically significant associations with tumor HPV-positivity and survival of SCCOP patients. Patients with AC/CC variant genotypes of MDM4 rs4245739 were approximately 3-fold more likely to be HPV16-positive tumors among SCCOP patients compared with common homozygous AA genotype (adjusted odds ratio = 3.2, 95% confidence interval = 1.9-5.5). Moreover, patients with MDM4 rs4245739 AC/CC variant genotypes had significantly better overall, disease-specific, and disease-free survival compared with those with the corresponding common homozygous AA genotype (all log-rank = P < .05); and these genotypes were significantly associated with an approximately three to four times reduced risk of overall death, death owing to disease, and recurrence after multivariable adjustment. Finally, the significant effects of MDM4 rs4245739 polymorphism on survival were found among HPV16-positive SCCOP patients only after the stratified analyses by tumor HPV status. We concluded that MDM4 rs4245739 polymorphism is significantly associated with tumor HPV status and survival of SCCOP, especially in HPV16-positive SCCOP patients treated with definitive radiotherapy; nevertheless, prospective larger studies are warranted.

In silico prediction of structural changes in\xa0human papillomavirus type 16 (HPV16) E6 oncoprotein and its variants

BackgroundHPV16 infection is one of the main risk factors involved in the development of cervical cancer, mainly due to the high oncogenic potential of the viral proteins E6 and E7, which are involved in the different processes of malignant transformation. There is a broad spectrum of intratypical variation of E6, which is reflected in its high diversity, biological behavior, global distribution and risk of causing cervical cancer. Experimental studies have shown that the intratypical variants of the protein E6 from the European variants (E-G350, E-A176/G350, E-C188/G350) and Asian-American variants (AAa and AAc), are capable of inducing the differential expression of genes involved in the development of cervical cancer.ResultsAn in silico analysis was performed to characterize the molecular effects of these variations using the structure of the HPV16 E6 oncoprotein (PDB: 4XR8; chain H) as a template. In particular, we evaluated the 3D structures of the intratypical variants by structural alignment, ERRAT, Ramachandran plots and prediction of protein disorder, which was further validated by molecular dynamics simulations. Our results, in general, showed no significant changes in the protein 3D structure. However, we observed subtle changes in protein physicochemical features and structural disorder in the N- and C-termini.ConclusionsOur results showed that mutations in the viral oncogene E6 of six high-risk HPV16 variants are effectively neutral and do not cause significant structural changes except slight variations of structural disorder. As structural disorder is involved in rewiring protein-protein interactions, these results suggest a differential pattern of interaction of E6 with the target protein P53 and possibly different patterns of tumor aggressiveness associated with certain types of variants of the E6 oncoprotein.

Identification of potential binding pocket on viral oncoprotein HPV16 E6: a promising anti-cancer target for small molecule drug discovery

BackgroundSeveral human cancers, especially cervical cancer are caused by the infection of high risk strains of human papillomaviruses (HPV), notably HPV16. It is implicated that the oncoprotein E6 expressed from HPV, is inhibiting the apoptotic pathway by binding to adaptor molecule FADD (Fas-associated death domain). Inhibiting E6 interactions with FADD could provide a promising treatment for cervical cancer. There are few small molecules reported to inhibit such interactions. However, the FADD binding site information on the HPV E6 is not currently available. This binding site information may provide an opportunity to design new small molecule inhibitors to treat E6 mediated cancers. In this study we report the possible binding pocket on HPV16 E6 oncoprotein by using activity data of reported inhibitors through a stepwise molecular modeling approach.ResultsBlind docking and removing duplicates followed by visual inspection to determine ligand-receptor interactions provided 68 possible binding sites on the E6 protein. Individual docking of all known inhibitors lead to the identification of 28 pockets having some kind of correlation with their activity data. It was also observed that several of these pockets overlapped with each other, having some amino acids in common. Amino acids Leu50 and Cys51 were identified as key E6 residues for high affinity ligand binding which are seen in most of these pockets. In most cases, ligands demonstrated a hydrogen bond interaction with Cys51. Ala61, Arg131 and Gln107 were also frequently observed showing interactions among these pockets. A few amino acids unique to each ligand were also identified representing additional interactions at the receptor site.ConclusionsAfter determining receptor-ligand interactions between E6 oncoprotein and the six known inhibitors, the amino acids Cys51, Leu50, Arg102, Arg131, Leu67, Val62, and Gln107 were identified to have importance in E6 inhibition. It was generally observed that Leu50 and Cys51 are necessary for high binding affinity with Cys51 being essential for hydrogen bonding. This study identified a potential binding pocket for the E6 inhibitors. Identification of the ligand binding pocket helps to design novel inhibitors of HPV16 E6 oncoprotein as a promising treatment for cervical cancer.

Therapeutic Vaccines Against Human Papilloma Viruses: Achievements and Prospects.

Human papillomaviruses of high carcinogenic risk (HR HPVs) are major etiological agents of malignant diseases of the cervix, vulva, penis, anal canal, larynx, head, and neck. Prophylactic vaccination against HPV, which mainly covers girls and women under 25, does not prevent vertical and horizontal HPV transmission in infants and children and does not have a therapeutic effect. As a result, a significant proportion of the population is not protected from the HPV infection and development of HPV-associated neoplastic transformation and cancer, which indicates the need for development and introduction of therapeutic HPV vaccines. Unlike prophylactic vaccines aimed at the formation of virus-neutralizing antibodies, therapeutic vaccines elicit cellular immune response leading to the elimination of infected and malignant cells expressing viral proteins. The ideal targets for vaccine immunotherapy are highly conserved HR HPV oncoproteins E6 and E7 expressed in precancerous and tumor tissues. Here, we describe expression of these proteins during different stages of HPV infection, their antigenic and immunogenic properties, and T-cell epitopes, the response to which correlates with natural regression of HPV-induced neoplastic changes. The review describes patterns of E6 and E7 oncoproteins presentation to the immune system as components of candidate vaccines along with the results of the most promising preclinical trials and animal models used in these trials. Special attention is paid to vaccine candidates which have shown efficacy in clinical trials in patients with HPV-associated neoplastic changes.

Therapeutic efficacy of a human papillomavirus type 16 E7 bacterial exotoxin fusion protein adjuvanted with CpG or GPI-0100 in a preclinical mouse model for HPV-associated disease

Persistent human papillomavirus (HPV) infection is causally linked to the development of several human cancers, including cervical, vulvar, vaginal, anal, penile, and oropharyngeal cancers. To address the need for a therapeutic vaccine against HPV-associated diseases, here we test and compare the immunogenicity and therapeutic efficacy of a bacterial exotoxin fusion protein covalently linked to the HPV16 E7 oncoprotein adjuvanted with CpG or GPI-0100 in the C3.43 preclinical HPV16-transformed tumor model. We show that TVGV-1 protein vaccine adjuvanted with either CpG or GPI-0100 adjuvant induces a high frequency of E7-specific CD8+ T cells, and both adjuvants are able to assist the immune response in inducing polyfunctional cytokine-secreting lytic T cells that show therapeutic efficacy against well-established C3.43 tumors. CpG-adjuvanted TVGV-1 resulted in higher frequencies of IFNγ secreting and degranulating E7-specific T cells compared to GPI-0100-adjuvanted TVGV-1, resulting in marginally increased in vivo efficacy. Despite minor differences in immune response outcomes, we consider both CpG ODN and GPI-0100 to be promising vaccine adjuvants to increase the immunogenicity and therapeutic efficacy of the TVGV-1 protein for HPV16-driven cancers.

Flax seed oil reduced tumor growth, modulated immune responses and decreased HPV E6 and E7 oncoprotein expression in a murine model of ectopic cervical cancer

Cervical cancer is the second leading cause of cancer death in women in India. Previously, we have reported that alpha linolenic acid (ALA), induced apoptosis in cervical cancer cell lines and reduced expression of E6 and E7 oncoproteins with simultaneous decrease in Cox2/VEGF/MAP kinase proteins. Here, we investigated the tumor retardation potential of flax oil (FO), rich in ALA, in mouse papilloma model. Flax oil significantly reduced tumor volume and weight in mice compared to the Tumor control (TC) group. Interestingly, compared to cisplatin (Cis) alone, there was slightly enhanced decrease in tumor weight when FO was given together with Cis (Cis + FO). A marked increase in plasma antioxidant levels in mice, and increase in lipid peroxidation in tumors with simultaneous decrease in liver tissues was observed in Cis + FO group compared to either TC or Cis groups. FO and Cis + FO significantly modulated immune response in mice by increasing CD8α and IFNγ and decreasing IL-4 expression. Interestingly, when given together with cisplatin, flax oil reduced HPV E6 and E7 oncoprotein expression with concomitant increase in the relative mRNA expression of tumor suppressor genes p53 and Rb. Thus, flax oil could be explored for its therapeutic potential in cervical cancer.

Optimization of peptide-based cancer vaccine compositions, by sequential screening, using versatile liposomal platform

Therapeutic cancer vaccines need thoughtful design to efficiently deliver appropriate antigens and adjuvants to the immune system. In the current study, we took advantage of the versatility of a liposomal platform to conceive and customize vaccines containing three elements needed for the induction of efficient antitumor immunity: i) a CD4 epitope peptide able to activate CD4+ T helper cells, ii) a CD8 tumor-specific epitope peptide recognized by CD8+ T cytotoxic cells and iii) Pattern Recognition Receptor (PRR) agonists which stand as adjuvants. Each type of component, conjugated to liposomes, was evaluated individually by comparing their vaccine efficacy after immunization of naïve mice. These screening steps resulted in the optimization of three liposomal constructs bearing a peptide from HA influenza virus protein as CD4 epitope, a peptide from HPV16 E7 oncoprotein as CD8 epitope and TLR4, TLR2/6 or NOD1 agonists as adjuvant, which displayed antitumor efficiency against a mouse model of disseminated tumors transformed by HPV16. Our results validated the interest of our customizable liposomal platform as delivery system for cancer vaccination. We also demonstrated its interest as tool for vaccine design allowing the strategical selection of components, and the evaluation of epitope-adjuvant association.

Long non-coding RNA FAM83H-AS1 is regulated by human papillomavirus 16 E6 independently of p53 in cervical cancer cells

High-risk human papillomavirus (HPV) infection is one of the first events in the process of carcinogenesis in cervical and head and neck cancers. The expression of the viral oncoproteins E6 and E7 are essential in this process by inactivating the tumor suppressor proteins p53 and Rb, respectively, in addition to their interactions with other host proteins. Non-coding RNAs, such as long non-coding RNAs (lncRNAs) have been found to be dysregulated in several cancers, suggesting an important role in tumorigenesis. In order to identify host lncRNAs affected by HPV infection, we expressed the high-risk HPV-16 E6 oncoprotein in primary human keratinocytes and measured the global lncRNA expression profile by high-throughput sequencing (RNA-seq). We found several host lncRNAs differentially expressed by E6 including GAS5, H19, and FAM83H-AS1. Interestingly, FAM83H-AS1 was found overexpressed in HPV-16 positive cervical cancer cell lines in an HPV-16 E6-dependent manner but independently of p53 regulation. Furthermore, FAM83H-AS1 was found to be regulated through the E6-p300 pathway. Knockdown of FAM83H-AS1 by siRNAs decreased cellular proliferation, migration and increased apoptosis. FAM83H-AS1 was also found to be altered in human cervical cancer tissues and high expression of this lncRNA was associated with worse overall survival, suggesting an important role in cervical carcinogenesis.