Understanding the Pattern of Oropharyngeal Cancers from North-East Romanian Patients

Background: Human papilloma virus (HPV) is acknowledged as a risk factor for oropharyngeal squamous cellular cancers (OPSCC), of which the dominant types are tonsillar (TSCC) and base of tongue cancer (BOTSCC). Objective: To assess the role of HPV in selected OPSCC cases, from Romanian patients by sensitive and complementary molecular assays. Material and Methods: Fifty-four formalin fixed paraffin embedded (FFPE) OPSCC samples were analyzed for HPV DNA by a PCR-based bead-based multiplex-assay. Thirty-four samples were tested for HPV RNA and for overexpression of p16INK4a by immunohistochemistry. Twenty samples were evaluated by Competitive Allele-Specific Taqman PCR (CAST-PCR) for fibroblast growth factor receptor 3 protein (FGFR3) status. Results: A total of 33.3% (18/54) OPSCC samples were positive for HPV DNA. HPV16 was the most frequent type (30%, 16/54); followed by HPV18 (3.7%, 2/54); and 1 sample (1.8%) was positive for both HPV16 and 18. HPV18 E6*I was detected in a HPV18 DNA-positive oropharynx tumor. Four samples positive for HPV16 were also positive for p16INK4a. All the tested samples were negative for FGFR3. Conclusions: The increased HPV16 prevalence is in line with similar studies and is a new confirmation that HPV16 is the most prevalent type in our country; supporting the potential benefit of prophylactic vaccines. Overall, there is no concordance between DNA and any of the two other analytes that are considered being markers of HPV-driven cancers. There is a need to explore novel screening strategies that could be broadly used in the clinical routine to initiate preventive measures.

Investigation of potential antiviral natural products with an effect on HPV18 E6 protein by molecular docking method

Background: Infection with the Human Papillomavirus (HPV) causes cellular dysplasia, which leads to cervical cancers in women and penile or rectal cancers in men. Objective: This in silico study identified the plant compounds with potential therapeutic effects against HPV 18 oncogenic virus using the molecular docking method. Methods: The three-dimensional (3D) structure of HPV18 E6 protein, as the target protein, and the 3D structure of plant compounds with potential therapeutic effect against viruses, as ligands, was obtained from the protein databases (RCSB) and PubChem, respectively. Both structures of ligands and target protein were subjected to AutoDock tools-1.5.6, ver.4 separately. The structure with the most negative affinity was docked to reconsider its connection location. The results were analyzed more based on pharmacodynamic and pharmacokinetic parameters. Results: The docking of HPV18 E6 protein with 19 selected ligands resulted in four compounds, curcumin, silymarin, saikosaponin c, and lactupicrin, showing the best docking scores; they had better binding free energies with HPV E6 protein. Among four compounds against HPV18 E6, silymarin and curcumin were less dangerous than other compounds due to the lack of inhibition of the human Ether-à-go-go-Related Gene (hERG). Of these two compounds, silymarin had lower oral absorption, lactopicrin had less skin absorption, lactopicrin is the substrate of P-gp, and saikosaponin c crosses the blood-brain barrier. Conclusion: Among potential antiviral plants against HPV18E6, four compounds were found to be effective. According to these findings, it is recommended that in vitro and in vivo examinations be conducted to determine the effectiveness of these compounds against HPV18 Keywords: Biological products, Antiviral agents, HPV18, Molecular docking, Computational biology, E6 protein

Antiproliferative Effects of AAV-Delivered CRISPR/Cas9-Based Degradation of the HPV18-E6 Gene in HeLa Cells

Human papillomavirus infections are associated with most cervical cancers, which are the fourth most common cancer in women. HPV-E6 protein binds to protein p53 and inhibits its function, leading to the switching of normal cells toward cancer cells. Here, we disrupted the HPV-E6 gene and investigated its effects on the proliferation and apoptosis of HeLa cells. The HPV18-E6 gene was targeted with two designed sgRNAs cloned into an AAV-CRISPR-based plasmid. The AAV-E6-CRISPR/Cas9 virions were prepared and titrated in HEK293t cells. The cleavage created in the HPV-E6 gene was detected using the T7E1 assay. Cell cycle profiling, MTT assay, and annexin V/PI staining were performed. Also, the p53 protein level was measured by Western blotting. Our data showed that disruption of the HPV-E6 gene led to increased cell apoptosis and decreased cell proliferation. A significant accumulation of infected cells in sub-G1 phase was observed in the cell profiling assay. Also, HPV-E6 gene disruption resulted in a significant increase in the level of P53 protein. Our findings indicated that AAV-mediated delivery of CRISPR/Cas9 can effectively target the HPV-E6 gene in HeLa cells, and its antiproliferative effects may provide therapeutic benefits of local administration of this gene-editing system for HPV-related cervical cancers.

Recognition of high-risk HPV E6 oncoproteins by 14-3-3 proteins studied by interactomics and crystallography

In tumors induced by high-risk mucosal human papillomaviruses (hrm-HPVs), HPV E6 oncoproteins inhibit apoptotic processes and sustain cell proliferation. E6 from all hrm-HPVs harbor a C-terminal short PDZ domain-binding motif (PBM), whose phosphorylation down-regulates PDZ binding but triggers E6 binding to 14-3-3 proteins. Here we classify PBMs of E6 proteins depending on their principle ability to be phosphorylated and subsequently acquire a 14-3-3-binding motif III consensus, (pS/pT)XX-COOH. Systematic competitive fluorescence polarization measurements show that the PBMs from four selected E6 oncoproteins bind all seven human 14-3-3 isoforms with distinct, wide-ranging affinities, obeying remarkable trends assigned to 14-3-3 isoform specificity and small E6 sequence variations. We crystallized the hrm-HPV18 E6 PBM bound to 14-3-3ζ, revealing a 14-3-3-motif III complex at 1.9 Å resolution. Using fluorescence polarization and crystallography, we also demonstrate that fusicoccin, a molecule that reinforces many known 14-3-3 complexes, destabilizes the 14-3-3-E6 interaction, indicating the druggability of that complex.

Recognition of high-risk HPV E6 oncoproteins by 14-3-3 proteins studied by interactomics and crystallography

In tumors induced by high-risk mucosal human papillomaviruses (hrm-HPVs), HPV E6 oncoproteins inhibit apoptotic processes and sustain cell proliferation. E6 from all hrm-HPVs harbor a C-terminal short PDZ domain-binding motif (PBM), whose phosphorylation down-regulates PDZ binding but triggers E6 binding to 14-3-3 proteins. Here we classify PBMs of E6 proteins depending on their principle ability to be phosphorylated and subsequently acquire a 14-3-3-binding motif III consensus, (pS/pT)XX-COOH. Systematic competitive fluorescence polarization measurements show that the PBMs from four selected E6 oncoproteins bind all seven human 14-3-3 isoforms with distinct, wide-ranging affinities, obeying remarkable trends assigned to 14-3-3 isoform specificity and small E6 sequence variations. We crystallized the hrm-HPV18 E6 PBM bound to 14-3-3ζ, revealing a 14-3-3-motif III complex at 1.9 Å resolution. Using fluorescence polarization and crystallography, we also demonstrate that fusicoccin, a molecule that reinforces many known 14-3-3 complexes, destabilizes the 14-3-3-E6 interaction, indicating the druggability of that complex.

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

ABSTRACTHuman papillomaviruses (HPVs) such as HPV16 and HPV18 can cause cancers of the cervix, vagina, vulva, penis, anus and oropharynx. Continuous expression of the HPV viral oncoproteins E6 and E7 are essential for transformation and maintenance of cancer cells. Therefore, therapeutic targeting of E6 and E7 genes can potentially be used to treat HPV-related cancers. Previous CRISPR/Cas9 studies on inactivation of E6 and E7 genes confirmed cell cycle arrest and apoptosis. Here we report that CRISPR/Cas9-based knockout of E6 and E7 can also trigger cellular senescence in HPV18 immortalized HeLa cells. Specifically, HeLa cells in which E6 and E7 were inactivated exhibited characteristic senescence markers like enlarged cell and nucleus surface area, increased β-galactosidase expression, and loss of lamin B1 with detection of cytoplasmic chromatin fragments. Furthermore, the knockout of HPV18 E6 and E7 proteins resulted in upregulation of p53/p21 and pRb/p21 levels in senescent cells. These senescent cells were devoid of characteristic apoptotic markers and re-introduction of codon-modified HPV18 E6 decreased p53 levels. Taken together, our study demonstrates that cellular senescence is as an alternative outcome of HPV oncogene inactivation by the CRISPR/Cas9 methodology.