E6 mRNA Research Articles

Overexpression of m6A-factors METTL3, ALKBH5, and YTHDC1 alters HPV16 mRNA splicing

We report that overexpression of the m6A-demethylase alkB homolog 5 RNA demethylase (ALKBH5) promoted production of intron retention on the human papillomavirus type 16 (HPV16) E6 mRNAs thereby promoting E6 mRNA production. ALKBH5 also altered alternative splicing of the late L1 mRNA by an exon skipping mechanism. Knock-down of ALKBH5 had the opposite effect on splicing of these HPV16 mRNAs. Overexpression of the m6A-methylase methyltransferase-like protein 3 (METLL3) induced production of intron-containing HPV16 E1 mRNAs over spliced E2 mRNAs and altered HPV16 L1 mRNA splicing in a manner opposite to ALKBH5. Overexpression of the nuclear m6A-“reader” YTH domain-containing protein 1 (YTHDC1), enhanced retention of the E6-encoding intron and promoted E6 mRNA production. We also show that HPV16 mRNAs are bound to YTHDC1 in human cells and that YTHDC1 affected splicing of HPV16 E6/E7 mRNAs produced from the episomal form of the HPV16 genome. Finally, we show that HPV16 mRNAs are m6A-methylated in tonsillar cancer cells. In summary, HPV16 mRNAs are methylated in HPV16-infected tonsillar cancer cells and overexpression of m6A-“writer” METTL3, m6A-“eraser” ALKBH5 and the m6A-“reader” YTHDC1 affected HPV16 mRNA splicing, suggesting that m6A plays an important role in the HPV16 gene expression program, at least in cancer cells.

Association of human papillomavirus in penile cancer: A single-center analysis.

ABSTRACTIntroduction:Human papillomavirus (HPV) is a known risk factor of penile cancer (PeCa). However, studies evaluating its true association are limited. In this study, we aimed to estimate HPV prevalence and its true association with PeCa in terms of molecular biological activities.Materials and Methods:This single-institutional prospective observational study was conducted between June 2016 and August 2019. We included 40 men with PeCa as a study group and 20 age-matched uncircumcised men who underwent circumcision for phimosis as a control group. Both the groups underwent deoxyribonucleic acid isolation for HPV subtyping followed by evaluation of relative E6/E7 messenger ribonucleic acid (mRNA) expression profile and relative telomerase activity in tissue samples. HPV-16 and -18 were categorized as high-risk, whereas HPV-6 and -11 were categorized as low-risk subtypes.Results:The mean (±standard deviation) age of PeCa was 51 ± 15.9 years. The majority of patients had stage II disease, and the most common procedure done was partial penectomy. The overall prevalence of HPV in PeCa was 42.5% (n = 17) as compared to 20% (n = 4) in controls. Among the subtypes, the most common subtype was HPV-16 noted in 33.3% (8/24) of cases, followed by HPV-18 in 29.2% (7/24) of cases. PeCa tissues had a significantly higher relative E7 mRNA expression for HPV-18 than the control group (P = 0.016). The mean relative telomerase activity was significantly higher in the PeCa tissues than the control group (138.66 vs. 14.46, P < 0.001). A significantly higher relative telomerase activity was noted in the PeCa tissues positive for high-risk HPV subtypes than controls (141.90 vs. 14.46, P = 0.0008), but not between high-risk HPV-positive and HPV-negative PeCa cases (141.90 vs. 137.03, P = 0.79). High-risk subtypes were not associated with tumor stage (P = 0.76) or lymph node metastasis (P = 0.816).Conclusions:HPV was associated in 42.5% of PeCa cases based on our experience from a single institution. PeCa tissues had a higher relative E7 mRNA expression for HPV-18 and relative telomerase activity as compared to controls suggesting their potential role as surrogate markers of virus-induced tumorigenesis.

Predominance of genomically defined A lineage of HPV16 over D lineage in Indian patients from eastern India with squamous cell carcinoma of the cervix in association with distinct oncogenic phenotypes

Human papillomavirus type-16 (HPV16) is classified into lineages, A, B, C and D and 10 sub-lineages portraying variable infectivity, persistence, and cytological outcomes, however, with geographical variations. Our objective was to delineate the distinctive features of lineages among cervical squamous cell carcinoma (SCC) in the eastern region of India. A total of 145 SCC cases and 24 non-malignant specimens, harboring episomal HPV16, were included. The presence of higher proportion of lineage A over D was observed among SCC cases (86.89% A1, 8.97% D1 and 4.14% D2), while only A1 sub-lineage viruses were found among control specimens. Among the A1 viruses, an association of variants in the E5 (Y44L, I65V), E6 (L83V) genes and LCR: C7577T with SCC, with combined Odd's ratio (95% CI) of 20.5(4.61–91.25) was observed. Network analyses revealed the presence of 10 clades of lineage A viruses comprising of 64 HPV16 genomes harboring the risk alleles. High episomal HPV16 DNA copy numbers (adjusted p-value= 0.0271) and E7 mRNA expression (p-value=0.000017) predominated in SCC with lineage A, over D. Our study highlights the distinctive modalities of oncogenicity among different HPV16 lineages.

Development of Antibodies against HPV-6 and HPV-11 for the Study of Laryngeal Papilloma.

Laryngeal papilloma (LP), which is associated with infection by human papillomavirus (HPV)-6 or -11, displays aggressive growth. The precise molecular mechanism underlying the tumorigenesis of LP has yet to be uncovered. Building on our earlier research into HPV-6, in this study, the viral gene expression of HPV-11 was investigated by quantitative PCR and DNA/RNA in situ hybridization. Additionally, newly developed antibodies against the E4 protein of HPV-6 and HPV-11 were evaluated by immunohistochemistry. The average viral load of HPV-11 in LP was 1.95 ± 0.66 × 105 copies/ng DNA, and 88% of HPV mRNA expression was found to be E4, E5a, and E5b mRNAs. According to RNA in situ hybridization, E4 and E5b mRNAs were expressed from the middle to upper part of the epithelium. E4 immunohistochemistry revealed a wide positive reaction in the upper cell layer in line with E4 mRNA expression. Other head and neck lesions with HPV-11 infection also showed a positive reaction in E4 immunohistochemistry. The distribution pattern of HPV DNA, viral mRNA, and E4 protein in LP with HPV-11 infection was quite similar to that of HPV-6. Therefore, it might be possible to apply these E4-specific antibodies in other functional studies as well as clinical applications, including targeted molecular therapies in patients with HPV-6 and HPV-11 infection.

Functional identification of ergothioneine transporter in rainbow trout (Oncorhynchus mykiss).

(2S)-3-(2-sulfanylidene-1,3-dihydroimidazol-4-yl)-2-(trimethylazaniumyl)propanoate (ergothioneine, ET) is hydrophilic antioxidant produced only by certain low-level organisms, but has been reported in the organs of some animals upon dietary intake, facilitated by the ergothioneine transporter (ETT). In this study, it was hypothesised that ETT is involved in the uptake of ET from diet in salmonids. The mechanism of ET uptake was evaluated through knockdown of candidate gene encoding ETT in a cell line (RTG-2) from rainbow trout (Oncorhynchus mykiss). ETT gene expression in blood cells and certain tissues from rainbow trout was also investigated as ET from the diet is expected to be carried over to the muscle. Knockdown of an ETT candidate (E1) in RTG-2 cells resulted in a remarkable decrease in E1 mRNA expression; uptake of ET was lower in the knocked group than in the control group suggesting that E1 functions as an ETT for rainbow trout. Furthermore, the expression of ETT gene in different tissues including the muscle points to ET uptake from the diet to the muscle of salmonids. In conclusion, this study partly reveals the possible pathway underlying ET uptake from the diet and its deposition in the muscle of edible fish species.

Usefulness of E7 mRNA in HPV16-Positive Women to Predict the Risk of Progression to HSIL/CIN2.

Objective: To evaluate whether E7 mRNA can predict the risk of progression in women with HPV16 infection. Design: A prospective observational study. Setting: A tertiary university hospital. Population: A cohort of 139 women referred to colposcopy for an abnormal screening result fulfilling the following inclusion criteria: (1) a positive test result confirming HPV16 infection; (2) a biopsy sample with a histological diagnosis of an absence of lesion or low-grade SIL/CIN grade1 (LSIL/CIN1); (3) no previous HPV vaccination; (4) no pregnancy; and (5) no previous cervical treatments; and (6) no immunosuppression. Methods: At the first visit, all women underwent a cervical sample for liquid-based cytology, HPV testing and genotyping, and HPV16 E7 mRNA analysis and a colposcopy with at least one colposcopy-guided biopsy. Follow-up visits were scheduled every six months. In each control, a liquid-based Pap smear, HPV testing, as well as a colposcopy examination with biopsy if necessary were performed. Main outcome measures: Histological diagnosis of HSIL/CIN2+ at any time during follow-up. Results: E7 mRNA expression was positive in 55/127 (43.3%) women included in the study and seven (12.7%) progressed to HSIL/CIN2+. In contrast, only 1/72 (1.4%) women with no HPV16 E7 mRNA expression progressed (p = 0.027). HPV16 E7 mRNA expression was associated with a 10-fold increased risk of progression (HR 10.0; 95% CI 1.2–81.4). Conclusions: HPV16 E7 mRNA could be useful for risk stratification of women with HPV16 infection in whom a HSIL/CIN2+ has been ruled out. Funding: Instituto de Salud Carlos III (ICSIII)-Fondo de Investigación Sanitaria and ERDF ‘One Way to Europe’ (PI17/00772).

Preliminary study of the use of E6/E7mRNA detection in screening and triage management of HR-HPV infection during pregnancy.

BackgroundDetection of E6 and E7 mRNA load of high-risk human papillomavirus (HR-HPV) infection during pregnancy was compared with conventional cytopathology and DNA detection by pathological examination as colposcopy to evaluate the application of E6 and E7 mRNA detection in the diagnosis and management in HR-HPV infection for high -grade cervical lesions during pregnancy.MethodsFrom January 2014 to June 2019, 1,058 pregnant women of childbearing age who were filed for regular obstetrics in the Department of Obstetrics and Gynecology, Xuanwu Hospital of Capital Medical University, were separately assessed using cervical liquid-based cytology and HPV DNA detection. If the results were abnormal, colposcopy was performed as a follow-up. The presence of HR-HPV E6/E7 mRNA fragments was detected through the HR-HPV E6/E7 mRNA test, and monitored at the same time as colposcopy. The diagnostic efficacy of the HR-HPV DNA test versus the HR-HPV E6/E7 mRNA test for high-grade cervical lesions during pregnancy was compared.ResultsThe positive rate of HR-HPV E6/E7 mRNA detection in the overall cervical intraepithelial neoplasia (CIN) and above during pregnancy was lower than that with HR-HPV DNA detection, and there was a significant statistical difference between the two methods. In CIN I and normal or inflammatory results, the positive rate of HR-HPV E6/E7 mRNA detection was lower than that of HR-HPV DNA detection, while in the results of CIN II and CIN III, the positive rate of the two was not significantly different. HR-HPV E6/E7 mRNA detection is the same as HR-HPV DNA detection, both of which increased with the severity of cervical lesions, and the positive rate increased. In cases of maintenance or progression of cervical lesions, the positive rate of HR-HPV E6/E7 mRNA detection during pregnancy can reach 81.8%. High-grade cervical lesions during pregnancy had a higher rate of reversal to a lower level after delivery.ConclusionsThe results suggested that the use of HR-HPV E6/E7 mRNA detection in cases of positive HR-HPV DNA detection can significantly improve the diagnostic specificity of CIN II and above high-grade cervical lesions.

Prognostic significance of human papillomavirus 16 viral load level in patients with oropharyngeal cancer.

Human papillomavirus (HPV) infection in patients with oropharyngeal squamous cell carcinoma (OPSCC) is a major determinant for better prognosis. However, there remain HPV‐positive patients who have poor outcomes. The stratification strategy for detecting high‐risk patients among those with HPV‐positive OPSCC has not been well delineated, especially for Asian patients. We undertook a retrospective cohort study on the survival rate of 89 Japanese patients diagnosed with primary OPSCC. The tumors were concurrently analyzed for the presence of HPV E6 DNA/mRNA, viral DNA load, p16 expression, viral physical status, and viral variant lineage. Human papillomavirus 16 viral DNA was found in 45 (51%) OPSCCs. Human papillomavirus 16 DNA‐positive OPSCCs with higher viral load (classified as HPV16 DNA‐medium/high OPSCCs) showed significantly favorable overall survival and progression‐free survival compared with HPV16 DNA‐positive OPSCCs with lower viral load (<10 copies/cell; HPV16 DNA‐low OPSCCs) and HPV16 DNA‐negative OPSCCs. E6 mRNA expression was observed in all HPV16 DNA‐medium/high OPSCCs but not in HPV16 DNA‐low OPSCCs. Notably, p16‐positive and HPV16 DNA‐negative/low OPSCCs showed significantly worse survival than p16‐positive and HPV16 DNA‐medium/high OPSCCs and resembled HPV‐unrelated OPSCCs with regard to survival and risk factor profile. Although not significant, a trend toward shorter survival was observed for HPV16‐integrated OPSCCs. Phylogenetic analysis revealed two major types of HPV16 variants termed Asian (A4) and European (A1/A2/A3) variants, but no difference in survival between these variants was observed. Altogether, these findings suggest that HPV viral load is a potentially informative factor for more accurate risk stratification of patients with OPSCC.

Diagnostic performance of HPV E6/E7 mRNA testing towards HPV‐DNA testing and p16/Ki67 immunostaining as a biomarker of high‐risk HPV recurrence in Greek women surgically treated for their cervical lesions

To evaluate the diagnostic performance of E6/E7 HPV-mRNA overexpression towards HPV-DNA testing and p16/Ki67 immunocytochemistry in a post-op population to verify if this biomarker can be effectively used as indicator of successful cervical intraepithelial neoplasia (CIN) treatment. Our study retrospectively analyzed 197 patients of our Colposcopy Clinic between January 2013 and September 2020 coming with an abnormal Pap smear suggestive for colposcopy, and after a series of follow-ups including liquid-based cytology (LBC) and punch-biopsy sampling, there were surgically treated. LBC was used for cytology and molecular analysis of the three HPV-related biomarkers. Six months after treatment, 93% of the HPV-mRNA-positive women became negative while this applied to only 80.2% of the HPV-DNA-positive women. HPV persistence was 6.9% at 6-12 months after treatment. The comparison among cytology, colposcopy, HPV-DNA test, and HPV-mRNA test after treatment revealed that the last one is the only with a strong correlation with actual severity (histology during treatment) (ρ=0.345, p=0.006) implying that clinical cases with more severe CIN may have higher chances of unsuccessful treatment. HPV-mRNA test had higher sensitivity (100%), specificity (96.88%), and positive predictive value (45.45%) for CIN2+ recurrent lesions when compared with HPV-DNA testing (80%, 82.81%, 10.81% respectively) and p16/Ki67 immunocytochemistry (80%, 95.83%, 33.33% respectively) while their negative predictive values were similar. E6/E7 mRNA detection has higher diagnostic values for the prediction of treatment failure compared with HPV-DNA testing and p16/Ki67 immunocytochemistry, and as an outcome could be used as predictive indicator of CIN-treatment status.

Oncolytic Adenovirus H101 Synergizes with Radiation in Cervical Cancer Cells.

A major challenge in cervical cancer radiotherapy is tailoring the radiation doses efficiently to eliminate malignant cells and reduce the side effects in normal tissues. Oncolytic adenovirus drug H101 was recently tested and approved as a topical adjuvant treatment for several malignancies. This study aimed to evaluate the potential neoadjuvant radiotherapy benefits of H101 by testing the inhibitory function of H101 in combination with radiation in different cervical cancer cells. Human cervical cancer cell lines C33a, SiHa, CaSki, and HeLa were treated with varying concentrations of H101 alone or in combination with radiation (2 Gy or 4 Gy). Cell viability and apoptosis were measured at the indicated time intervals. HPV16 E6 and cellular p53 mRNA expression alteration was measured by qRT-PCR. In situ RNA scope was used to determine HPV E6 status. P53 protein alterations were detected by Western blot. Cell viability and apoptosis assays revealed that the combination of a high dose of H101 (MOI=1000, 10000) with radiation yielded a synergistic anticancer effect in all tested cervical cancer cell lines (P<0.05), with the greatest effect achieved in HPV-negative C33a cells (P<0.05). Low-HPV16-viral-load SiHa cells were more sensitive to the combination therapy than high-HPV16- viral-load CaSki cells (P<0.05). The combined treatment reduced HPV16 E6 expression and increased cellular P53 levels compared to those observed with radiation alone in SiHa and CaSki cells (P<0.05). Oncolytic adenovirus H101 effectively enhances the antitumor efficacy of radiation in cervical cancer cells and may serve as a novel combination therapy for cervical cancer.

LncRNA HOTAIR promotes proliferation and inhibits apoptosis by sponging miR-214-3p in HPV16 positive cervical cancer cells

BackgroundCervical cancer (CC) is one of the most common gynaecological malignancies all around the world. The mechanisms of cervical carcinoma formation remain under close scrutiny. The long non-coding RNAs (lncRNA) and microRNAs (miRNAs) play important roles in controlling gene expression and promoting the development and progression of cervical cancer by acting as competitive endogenous RNA (ceRNA). However, the roles of lncRNA associated with ceRNAs in cervical carcinogenesis remains unknown. In this study, the expression of long non-coding RNA HOTAIR was investigated in HPV16 positive cervical cancer cells, the candidate miRNAs and target genes were identified to clarify putative ceRNAs of HOTAIR/miRNA in cervical cancer cells.MethodsThe proliferation ability of cells was measured by CCK8 and EdU incorporation assays and cell apoptosis was analyzed by flow cytometry. The expression of HOTAIR, miR-214-3p, HPV16 E7 mRNA were detected by qRT-PCR. As for searching for the interaction between miR-214-3p and HOTAIR, the binding sites for miR-214-3p on HOTAIR was predicted by starbase v2.0 database, then dual-luciferase assay was used to verify the binding sites. In addition, Gene Ontology (GO) and protein–protein interaction (PPI) network analysis of target genes of miR-214-3p were performed with bioinformatics analysis. The potential signal pathway regulated by HOTAIR/miR-214-3p was predicted by KEGG enrichment analysis and confirmed by qPCR and WB analysis in cervical cancer cells.ResultsOur results showed that expression of HOTAIR was up-regulated, while that of miR-214-3p was down-regulated in HPV16-positive cervical cancer cells. The expression status of HPV16 E7 played an important role in regulating expression of HOTAIR or miR-214-3p in cervical cancer cells. HOTAIR knockdown could significantly inhibited cell proliferate ability and promote cellular apoptosis, whereas the inhibition of miR-214-3p expression partially reversed such results. Bioinformatics analysis identified 1451 genes as target genes of miR-214-3p. The Gene ontology (GO) and KEGG Pathway enrichment analysis showed that these target genes were mainly related to regulation of cell communication, protein binding, enzyme binding and transferase activity, and Wnt ligand biogenesis. Pathway enrichment analysis results showed that the predicted target genes were significantly enriched in Wnt/β-catenin signaling pathway. Finally, our results confirmed that miR-214-3p could significantly inhibit β-catenin expression in HPV16 positive cancer cells by qPCR and WB analysis.ConclusionHOTAIR could act as a ceRNA through binding to miR-214-3p, promote cell proliferation and inhibit the apoptosis of HPV16 positive cervical cancer. HOTAIR/miR-214-3p/Wnt/β-catenin signal pathway might played important regulated roles in HPV16 positive cervical cancer. Our results provided new insight into defining novel biomarkers for cervical cancer.

Molecular detection and seroprevalence of classical swine fever virus from 2016 to 2018 in pigs of Mizoram, India

Classical swine fever (CSF) is a fatal endemic disease of pig population of North eastern India in particular and India in general. Present study revealed molecular detection of CSFV and seroprevalence of the disease in pig population of Mizoram, India during 2016–2018. Serum samples from apparently healthy, unvaccinated pigs were collected in collaboration with the State Animal Husbandry and Veterinary Department, Mizoram and a total of 594 serum samples from 7 districts were subjected to detection of CSFV specific antibodies by indirect ELISA. A total of 206 (34.68%) serum samples were positive for CSFV antibodies by ELISA. District wise, Saiha district showed highest seroprevalence of the disease followed by Kolasib and Serchhip. Apart from this, during the same time period, CSFV suspected samples received in the Department of Veterinary Microbiology consisting of 269 serum samples, 10 whole blood and 83 tissue samples obtained from 8 districts of Mizoram were subjected to detection of NS5b and E2 mRNA transcripts by nRT-PCR of which a total of 42 (11.60%) samples including serum (5.58%), tissues (27.71%) and whole blood (40%) were positive for the NS5b and E2 mRNA transcripts, specific for CSFV. District wise analysis revealed that Aizawl has the highest percentage of positive samples of CSFV followed by Saiha and Lawngtlai district.

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.

Enhancing immunogenicity of HPV16 E7 DNA vaccine by conjugating codon-optimized GM-CSF to HPV16 E7 DNA

ObjectiveTo generate immunity against human papillomavirus (HPV), the use of a recombinant DNA vaccine to carry an appropriate target gene is a promising and cost-effective approach. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent immunomodulatory cytokine that enhances the efficacy of vaccines by promoting the development and prolongation of humoral and cellular immunity. In this study, we linked codon-optimized GM-CSF (cGM-CSF) to the HPV16 E7 sequence as fused protein and evaluated the immunogenic potential of this DNA vaccine. Materials and methodsWe have demonstrated that cGM-CSF enhanced immunity against tumor challenges by generating and promoting the proliferation of HPV16 E7-specific CD8+ T cells, which secrete IFN-γ in the murine model. In this study, we aimed to evaluate the immunogenic potential of DNA vaccine that constructed by linking codon-optimized GM-CSF to HPV16 E7 sequence in the animal model. We study the half-life of RNA decay and cellular location of HPV16 E7 by Q-PCR and Western blot. We also assess immune response in the animal model by flow cytometry and ELISA. ResultsThe cGM–CSF–E7 sequence increased and extended the expression of E7 mRNA, in comparison with the E7 sequence alone. Mice vaccinated with the cGM–CSF–E7 DNA vaccine exhibited a slower rate of tumor growth than those vaccinated with the unconjugated E7 DNA vaccine. We also found that the CD4 and CD8+ T cells from these mice showed strong secretion of IFN-γ. ConclusionThrough in vivo antibody depletion experiments, we demonstrated that both CD4+ and CD8+ T cells play an important role in the suppression of tumor growth.

Viral and cellular biomarkers used for improving prevention and management of cervical cancer

Cervical cancer remains one of the preventable cause of women’s death, the second most common cause of death in Europe. Several screening tests for precancerous lesions are available, including cervicovaginal cytology and screening of high-risk human papillomavirus (HPV) strains – 16, 18, 31, 33. Cervical cytology (Babeş-Papanicolau smear test) remains the elected method for cervical cancer screening in many countries. Last decades, HPV DNA sequence was identified, including gene number and encoded proteins. HPV DNA gene expression involves the use of multiple promoters and complex patterns of splicing, which served as a model for the elaboration of cellular and molecular biomarkers for the identification. Herein, we present the usefulness and efficiency of viral and cellular biomarkers in improving the diagnosis and managing precursor lesions of cervical cancer. Viral and cellular biomarkers can help differentiating low-grade lesion leading to a better diagnosis. HPV DNA tests, although a primary screening method in actual guidelines, can serve as a better screening test method. Other viral biomarkers that include E6 and E7 mRNA testing and determining the methylation of viral genes have good test screening results. Cellular biomarkers such as the immunocytochemical staining of p16INK4a, alone or combined with the proliferation marker Ki-67, can reveal anomalies in the cell cycle. The E4 protein, strongly expressed in productive infections, can prove a transforming infection when it is absent. In recent years, detection of the TERC gene and oncogenetic microRNAs expressed early in carcinogenesis before clinical findings show great promise in differentiating lesions likely to progress to cancer. In conclusion, biomarkers are more powerful instruments to enhance early pre-cervical cancer lesions diagnosis, which allow better prognosis and more reliable treatment.

Letter to “The application of PAX1 methylation detection and HPV E6/E7 mRNA detection in cervical cancer screening”

Letter to “The application of <scp>PAX1</scp> methylation detection and <scp>HPV E6</scp>/<scp>E7 mRNA</scp> detection in cervical cancer screening”

Full-genome characterization of a novel Felis catus papillomavirus 4 subtype identified in a cutaneous squamous cell carcinoma of a domestic cat.

The present study describes two full-genome sequences of Felis catus papillomavirus type 4 (FcaPV4) identified in squamous cell carcinoma (SCC) of two domestic cats. Two full-genome sequences of FcaPV4 were detected and characterized by PCR and sequencing. The L1 nucleotide sequence homology of one case showed 95.70% sequence identity to the reference FcaPV4, suggesting that this isolate should be classified as a subtype. Reverse-transcriptase PCR (RT-PCR) of two oncogenes, E6 and E7 was performed to confirm mRNA expression. Expression of E6 and E7 mRNA was detected in both cases, suggesting that FcaPV4 contributes to the development of SCC. This is the first report of FcaPV4 subtype. The present study will update the genomic features of FcaPV4 and contribute to deepening our knowledge about the etiological roles of FcaPV4 in feline cutaneous SCC.

Abstract PO-013: Irradiation induced expression of cytosolic DNA sensors and cytokines is associated with HPV status of pharyngeal squamous cell carcinoma cells

Abstract Human papillomavirus (HPV) positive pharyngeal squamous cell carcinoma (PSCC) appears to differ from HPV negative PSCC regarding several different characteristics due to the expression of HPV viral oncoproteins E6 and E7. Clinical studies suggest that HPV positive PSCC are more radiosensitive than HPV negative ones, however the exact mechanisms are still not known. It was previously shown that after X-ray radiation DNA accumulates in the cytosol of survived cells and can be sensed by specific pattern recognition receptors, i.e. cytosolic DNA sensors, which can modulate immune response and thus radiosensitivity of tumors. Therefore, the aim of our study was first to investigate the radiosensitivity and involvement of DNA sensing pathways in response to radiation in HPV positive and HPV negative PSCC in vitro. For determination of the radiosensitivity, HPV negative (FaDu and UM-SCC-6) and HPV positive (2A3 and UPCI:SCC090) PSCC cell lines were plated in 96 well plates and irradiated with different single doses (0, 2, 4, 6, 8 and 10 Gy). After 4 cell’s division PrestoBlue™ Cell Viability Reagent was used to determine the survival of cells taking into account different growth rate of the cells. For determination of DNA sensors and cytokines mRNA levels, PSCC cell lines were plated and irradiated with different single doses (0, 4 and 8 Gy). After 24 and 48 hours RNA was isolated using peqGOLD Total RNA Kit (VWR, West Chester, PA, USA) and reverse transcribed into cDNA using the SuperScript VILO cDNA Synthesis Kit (Invitrogen, Thermo Fisher Scientific). qRT-PCR was used to determine viral load (expression of HPV oncoproteins E6 and E7 mRNAs), expression of DNA sensors (cGAS, STING, RIG-I, DAI, IFI16, DDX60) and cytokines (IFNb, TNFa, IL1b). The expression of cytosolic DNA sensors and cytokines was dose- and time-dependent in both types of cells. The expression of DNA sensors DAI and RIG-I after irradiation was upregulated in HPV negative cell lines but not in HPV positive ones. The expression of cytokines IFN β and IL 1B was also upregulated only in HPV negative cell lines. Between the two HPV negative cell lines FaDu and SCC6, the expression profile did not differ, while the expression of DNA sensors and cytokines was higher in HPV positive cell line 2A3 compared to UM-SCC-6 cell lines, which has a higher level of viral proteins E6 and E7. In conclusion, our results indicate that on the in vitro level, DNA sensors are differentially expressed in response to irradiation in PSCC cell lines based on HPV infection but does not affect the radiosensitivity of the cells. Further in vivo studies are needed to elucidate the involvement of immune system in the response. Citation Format: Kristina Levpuscek, Tanja Jesenko, Primoz Strojan, Gregor Sersa, Maja Cemazar. Irradiation induced expression of cytosolic DNA sensors and cytokines is associated with HPV status of pharyngeal squamous cell carcinoma cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-013.

The Major Constituent of Green Tea, Epigallocatechin-3-Gallate (EGCG), Inhibits the Growth of HPV18-Infected Keratinocytes by Stimulating Proteasomal Turnover of the E6 and E7 Oncoproteins.

Epigallocatechin-3-gallate (EGCG), the primary bioactive polyphenol in green tea, has been shown to inhibit the growth of human papilloma virus (HPV)-transformed keratinocytes. Here, we set out to examine the consequences of EGCG treatment on the growth of HPV18-immortalised foreskin keratinocytes (HFK-HPV18) and an authentic HPV18-positive vulvar intraepithelial neoplasia (VIN) clone, focusing on its ability to influence cell proliferation and differentiation and to impact on viral oncogene expression and virus replication. EGCG treatment was associated with degradation of the E6 and E7 oncoproteins and an upregulation of their associated tumour suppressor genes; consequently, keratinocyte proliferation was inhibited in both monolayer and organotypic raft culture. While EGCG exerted a profound effect on cell proliferation, it had little impact on keratinocyte differentiation. Expression of the late viral protein E4 was suppressed in the presence of EGCG, suggesting that EGCG was able to block productive viral replication in differentiating keratinocytes. Although EGCG did not alter the levels of E6 and E7 mRNA, it enhanced the turnover of the E6 and E7 proteins. The addition of MG132, a proteasome inhibitor, to EGCG-treated keratinocytes led to the accumulation of the E6/E7 proteins, showing that EGCG acts as an anti-viral, targeting the E6 and E7 proteins for proteasome-mediated degradation.

Coordinated Expression of HPV-6 Genes with Predominant E4 and E5 Expression in Laryngeal Papilloma.

Laryngeal papilloma (LP) associated with human papillomavirus (HPV)-6 or -11 infection shows aggressive growth. However, the detailed molecular mechanism of virus-driven tumorigenesis has not been uncovered fully. HPV-6 viral gene expression and dynamic alterations were investigated with in situ localization of viral DNA and RNA in 13 patients with HPV-6-infected laryngeal papilloma. The average viral load was 4.80 × 105 ± 1.86 × 105 copies/ng DNA. E4, E5a, and E5b mRNAs accounted for 96% of the expression of 9 mRNAs. The alteration of viral DNA load during recurrence paralleled the mRNA expression levels, and the expression of all mRNAs showed a similar curve. E4, E5a, and E5b were expressed in the middle to upper part of the epithelium and were co-expressed in the same cells. E4 immunohistochemistry demonstrated an extensively positive reaction in the upper cell layer in accordance with E4 mRNA expression. These results suggest that individual viral genes are coordinately expressed for viral replication, virus release, and immunosurveillance avoidance. The newly developed E4-specific monoclonal antibody can be applied to further functional studies and clinical applications such as targeted molecular therapies.