HPV16 E6 Gene Research Articles

Recombinant adenoviruses expressing HPV16/18 E7 upregulate the HDAC6 and DNMT3B genes in C33A cells.

High-risk human papillomavirus (HPV) is a carcinogenic virus associated with nearly all cases of cervical cancer, as well as an increasing number of anal and oral cancers. The two carcinogenic proteins of HPV, E6 and E7, can immortalize keratinocytes and are essential for HPV-related cellular transformation. Currently, the global regulatory effects of these oncogenic proteins on the host proteome are not fully understood, and further exploration of the functions and carcinogenic mechanisms of E6 and E7 proteins is needed. We used a previously established platform in our laboratory for constructing recombinant adenoviral plasmids expressing the HPV16 E7 gene to further construct recombinant virus particles expressing HPV16/18 E6, E7, and both E6 and E7 genes. These recombinant viruses were used to infect C33A cells to achieve sustained expression of the HPV16/18 E6/E7 genes. Subsequently, total RNA was extracted and RNA-Seq technology was employed for transcriptome sequencing to identify differentially expressed genes associated with HPV infection in cervical cancer. RNA-Seq analysis revealed that overexpression of the HPV16/18 E6/E7 genes upregulated GP6, CD36, HDAC6, ESPL1, and DNMT3B among the differentially expressed genes (DEGs) associated with cervical cancer. Spearman correlation analysis revealed a statistically significant correlation between the HDAC6 and DNMT3B genes and key pathways, including DNA replication, tumor proliferation signature, G2M checkpoint, p53 pathways, and PI3K/AKT/mTOR signaling pathways. Further, qRT-PCR and Western blot analyses indicated that both HPV16/18 E7 can upregulate the expression of HDAC6 and DNMT3B, genes associated with HPV infection-related cervical cancer. The successful expression of HPV16/18 E6/E7 in cells indicates that the recombinant viruses retain the replication and infection capabilities of Ad4. Furthermore, the recombinant viruses expressing HPV16/18 E7 can upregulate the HDAC6 and DNMT3B genes involved in cervical cancer pathways, thereby influencing the cell cycle. Additionally, HDAC6 and DNMT3B are emerging as important therapeutic targets for cancer. This study lays the foundation for further exploration of the oncogenic mechanisms of HPV E6/E7 and may provide new directions for the treatment of HPV-related cancers.

The difference of transcriptome of HPV-infected patients contributes more to the occurrence of cervical cancer than the mutations of E6 and E7 genes in HPV16.

Human papillomavirus (HPV) E6 and E7 genes are biomarkers and drivers of the progression of cervical cancer (CxCa). The aim of this study was to investigate the relationship between HPV16 E6, E7 gene mutations and the occurrence and development of CxCa. Cervical exfoliated cells and clinical data of patients with cervical diseases were collected. Sample DNA was extracted, the E6 and E7 gene fragments were amplified by PCR, and the mutations were detected by Sanger sequencing and compared with standard sequences. Microarray was used to sequence the transcriptome of cells. Data of transcriptome analyzed and visualized using R software and its packages. Analysis of clinical characteristics demonstrated the association of HPV16 infection with CxCa (P < .05). Sanger sequencing results showed that the mutation sites of E6 gene included T178G/A, T350G, A131C, and T241G; among these, A131C and T241G were synonymous mutations. The mutation sites of E7 gene included A647G, T846C, G666A, T843C, and T760C, and all of them were synonymous mutations except A647G. There was no significant difference in the distribution of HPV16 E6, E7 mutations among CxCa, cervical intraepithelial neoplasia, and infection groups (P > .05). Compared with the non- CxCa group, gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes (DEGs) showed more significant enrichment of DEGs in the biological processes, pathways, and diseases closely related to cancer. Compared with the non-mutation group, the DEGs in the E6, E7 gene mutation group were significantly enriched in the events related to infection and immunity. To summarize, HPV16 may be associated with the occurrence and development of CxCa, but HPV16 E6 and E7 gene mutations have little effect on the occurrence and development of CxCa. Individual differences may have a greater effect on the progression of CxCa.

Deciphering the divergent transcriptomic landscapes of cervical cancer cells grown in 3D and 2D cell culture systems.

Cervical cancer remains a significant health challenge for women worldwide, with a disproportionate impact on developing regions like sub-Saharan Africa. Taking advantage of recent advancements in developing suitable preclinical models to study cell proliferation, differentiation, and gene expression, we used RNA sequencing to compare the transcriptomic profiles of SiHa cervical cancer cells grown in 3D versus 2D culture systems. Pathway analysis of 3D cultures revealed upregulation of immune activation, angiogenesis, and tissue remodeling pathways. The high expression of cytokines, chemokines, matrix metalloproteinases, and immediate early genes, suggests that 3D cultures replicate the tumor microenvironment better than 2D monolayer cultures. HPV gene expression analysis further demonstrated higher expression levels of HPV16 E1, E2, E6, and E7 genes in 3D versus 2D cultures. Further, by using a set of linear models, we identified 79 significantly differentially expressed genes in 3D culture compared to 2D culture conditions, independent of HPV16 viral gene effects. We subsequently validated five of these genes at the protein level in both the SiHa cell line and a newly developed, patient-derived cervical cancer cell line. In addition, correlation analysis identified 26 human genes positively correlated with viral genes across 2D and 3D culture conditions. The top five 3D versus 2D differentially expressed and HPV-correlated genes were validated via qRT-PCR in our patient derived cell line. Altogether, these findings suggest that 3D cultures provide superior model systems to explore mechanisms of immune evasion, cancer progression and antiviral therapeutics.

TRANSLACOL project: Nodal human papillomavirus tumoral DNA detection by ddPCR for survival prediction in early cervical cancer patients without pelvic lymph node invasion

In early cervical cancer (EEC), 10 to 15% of patients without nodal metastasis (N-) will suffer from recurrences with further similar survival as N+ patients. However, no clinical, imaging or pathological risk-factor is today available to identify them. In the present study, we hypothesized that the N- histologically characterized patients who present a poor prognosis could be patients for whom metastasis are missed by classical procedure. Therefore, we propose to research HPV tumoral DNA (HPVtDNA) in pelvic Sentinel Lymph Nodes (SLN) biopsy using ultrasensitive droplet-based digital PCR (ddPCR) to detect eventual occult metastasis. Sixty HPV16, HPV18 or HPV33 positive EEC N- patients with available SLN were included. In SLN, HPV16 E6, HPV18 E7 and HPV33 E6 gene were respectively detected using ultrasensitive ddPCR technology. Survival data were analysed using Kaplan-Meier-curves and log-rank-test to compare progression-free survival (PFS) and disease-specific survival (DSS) in two groups according to their HPVtDNA status in SLN. More than half (51.7%) of the patients finally showed HPVtDNA positivity in SLN initially diagnosed as negative by histology. Two patients with negative HPVtDNA SLN and 6 with positive HPVtDNA SLN group presented recurrence. Finally, all of the 4 deaths listed in our study occurred in the positive HPVtDNA SLN group. These observations hint that the use of ultrasensitive ddPCR to detect HPVtDNA in SLN could allow the identification of two subgroups of histologically N- patients that may have different prognosis and outcome. To our knowledge, our study is the first one to evaluate the detection of HPVtDNA in SLN in early cervical cancer using ddPCR highlighting its interest as a complementary tool for N- specific early cervical cancer diagnosis.

Genotypic distribution of human papillomavirus and phylogenetic analysis of E6 and E7 gene of HR-HPV variants isolated from Pakistani population

High-risk-human papillomavirus (HR-HPV)-induced cervical cancer is the second most common cause of death among females worldwide. HPV16 is the most prevalent HR-HPV infection worldwide. This study found the genotypic distribution of HR-HPV in the local population and investigated the sequence variations among the E6 and E7 oncogenes of the local HPV16 genotype to the E6 and E7 oncogenes of the foreign HPV16 genotypes and constructed a phylogenetic relationship based on nucleotide sequence comparison among the variants identified in our study along with previously reported isolates that were obtained from different regions of the world. The samples were collected from patients with cervical cancer. Genomic DNA was extracted, and HR-HPV genotypes were determined using real-time PCR. The HPV16 E6 and E7 genes were amplified and sequenced. A HPV16 phylogenetic tree was constructed using the maximum likelihood method with MEGA 7. HPV16 was the most prevalent human papillomavirus (HPV) type identified in the present study. HPV16 isolates belonged to the A1 sublineage of the European branch. Twenty-one nucleotide sequences were included in this analysis. The first, second, and third codon positions are also included. The final dataset included 776 positions.

Genetic Variability in the E6, E7, and L1 Genes of Human Papillomavirus Types 16 and 18 among Women in Saudi Arabia.

Cervical cancer is the eighth most frequent cancer in Saudi Arabia, and most cases are associated with human papillomavirus (HPV) types 16 and 18. HPV-induced carcinogenesis may be associated with the intra-type variant, genetic mutation, or the continuous expression of viral oncogenes E6 and E7. Infection efficiency and virus antigenicity may be affected by changes in the L1 gene. Thus, this retrospective cohort study analyzed E6, E7, and L1 gene mutations in cervical specimens collected from Saudi women positive for HPV16 or HPV18 infection. HPV16 and HPV18 lineages in these specimens were predominantly from Europe. The L83V mutation in the E6 gene of HPV16 showed sufficient oncogenic potential for progression to cervical cancer. By contrast, the L28F mutation in the E7 gene of HPV16 was associated with a low risk of cervical cancer. Other specific HPV16 and HPV18 mutations were associated with an increased risk of cancer, cancer progression, viral load, and age. Four novel mutations, K53T, K53N, R365P, and K443N, were identified in the L1 gene of HPV16. These findings for HPV16 and HPV18 lineages and mutations in the E6, E7, and L1 genes among women in Saudi Arabia may inform the design and development of effective molecular diagnostic tests and vaccination strategies for the Saudi population.

HPV16 E7 Nucleotide Variants Found in Cancer-Free Subjects Affect E7 Protein Expression and Transformation.

Simple SummaryHuman papillomaviruses (HPV) are one of the deadliest viruses causing cancer resulting in over 300,000 deaths annually in the world. HPV16 is the most oncogenic form of HPV and is responsible for over half of all HPV-driven tumors. The viral E7 gene encodes a powerful oncoprotein that can cause infected cells to have uncontrolled growth. A previous study showed that HPV16 isolates with amino acid alterations in the E7 gene were nearly always found in women with HPV infection but no cancer. To study the role of these variants, we expressed them in cultured cells. Most variants produced lower levels of protein, and selected variants had reduced activity in allowing cells to grow in assays that measure tumor cell invasiveness. The data indicate that inhibition of E7 could be a practical approach to treating HPV-driven cancers.The human papillomavirus (HPV) type 16 E7 oncogene is critical to carcinogenesis and highly conserved. Previous studies identified a preponderance of non-synonymous E7 variants amongst HPV16-positive cancer-free controls compared to those with cervical cancer. To investigate the function of E7 variants, we constructed full-length HPV16 E7 genes and tested variants at positions H9R, D21N, N29S, E33K, T56I, D62N, S63F, S63P, T64M, E80K, D81N, P92L, and P92S (found only in controls); D14E, N29H cervical intraepithelial neoplasia (CIN2), and P6L, H51N, R77S (CIN3). We determined the steady-state level of cytoplasmic and nuclear HPV16 E7 protein. All variants from controls showed a reduced level of E7 protein, with 7/13 variants having lower protein levels. In contrast, 2/3 variants from the CIN3 precancer group had near-wild type E7 levels. We assayed the activity of representative variants in stably transfected NIH3T3 cells. The H9R, E33K, P92L, and P92S variants found in control subjects had lower transforming activity than D14E and N29H variants (CIN2), and the R77S (CIN3) had activity only slightly reduced from wild-type E7. In addition, R77S and WT E7 caused increased migration of NIH3T3 cells in a wound-healing assay compared with H9R, E33K, P92L, and P92S (controls) and D14E (CIN2). These data provide evidence that the E7 variants found in HPV16-positive cancer-free women are partially defective for transformation and cell migration, further demonstrating the importance of fully active E7 in cancer development.

The T350G Variation of Human Papillomavirus 16 E6 Gene Prevails in Oropharyngeal Cancer from a Small Cohort of Greek Patients.

Recent trends have shown a dramatic rise in the incidence of oropharyngeal squamous cell carcinoma strongly associated with high-risk human papillomavirus (HPV) of type 16. The genetic variability of HPV16 has been extensively studied in cervical cancer but there are very limited published data concerning the genetic variations of this HPV type in oropharyngeal cancer. In the present study, the genetic variations of HPV16 E6 gene sequences originated from a small cohort of Greek patients diagnosed with oropharyngeal cancer were assessed. The vast majority of the sequences clustered within the European variant branch. The T350G variation was found to be the predominant one. This finding may indicate the need for further studies that could explain the possible impact of this variant in the pathomechanisms of oropharyngeal cancer.

Determination of Human Papillomavirus Type 18 Lineage of E6: A Population Study from Iran.

The epidemiological studies in Iran on HPV18 nucleotide changes are rare. This type of virus is prevalent in the Iranian population. Therefore, in the present study, we aimed to identify the genetic variability in HPV18 in the E6 region to evaluate the prevalence of lineage distribution and sublineages in a sample population in Iran. Overall, 60 HPV18 confirmed cases were investigated between 2019 and 2021. The specimens were collected, and molecular genotyping was done using the Linear Array HPV Genotyping Test. DNA extraction was performed by a viral DNA/RNA kit. The HPV E6 gene was amplified by using type-specific primers designed according to the HPV18 genome prototype sequence. The sequencing of the E6 region was successfully done on 43 samples which were then compared to the reference sequence. The most frequent sublineage of HPV18 in this study was A4 (69.7%), followed by A1 (18.6%) and A3 (11.6%). Neither A2 nor A5 sublineage was not detected in this study. The related nucleotide acid changes according to the main references were as follows: A3: T104C/T232G/T485C/C549A, A4: T104C/T485C/C549A. The predominance of A lineage with the high frequency of A4 sublineage was found in the present research. The importance of sublineages in susceptibility to a progressive form of infection requires to be more investigated among the different population.

HPV16 E6 gene polymorphisms and the functions of the mutation site in cervical cancer among Uygur ethnic and Han nationality women in Xinjiang, China

BackgroundTo investigate the genotype distribution of human papillomavirus (HPV) in infected Uygur and Han women in Xinjiang, China; analyze the HPV16 E6 gene polymorphism site and relationship with the development of cervical cancer.MethodsThe HPV16 E6 sequence was analyzed using the European standard prototype to perform an evolutionary tree. HPV16 E6-T295/T350, G295/G350, and T295/G350 GV230 vectors were stably transfected into cervical cancer C33A cells to analyze the cell proliferation, migration and invasion, apoptosis by CCK8 and clonogenic assays, transwell and cell scratch assays, FACS experiments.ResultsThe total HPV infection rate was 26.390% (760/2879), whereas the Uygur 22.87% (196/857) and the Han was 27.89% (564/2022) (P < 0.05). Among 110 mutations, 65 cases of E6 genes were mutated at nucleotide 350 (T350G) with the leucine changing to valine (L83V). Moreover, there were 7 cases of E6 gene mutated at nucleotide 295 (T295G) with aspartic changing to glutamic (D64E). When E6 vector(s) of mutations sites were transfected into C33A cells, they were found to promote cellular proliferation, migration, invasion, and inhibit apoptosis. T295/G350-E6 was significantly stronger than G295/G350 and T295/T350, G295/G350 was significantly stronger than T295/T350 (P < 0.05). The T295/G350 had the strongest effect on C33A cells and G295/G350 was significantly stronger than T295/T350 (P < 0.05).ConclusionsThe positive HPV infection rates differed between the Uygur and Han in Xinjiang, China, and the genotype distribution of infection was different. After transfecting C33A cells with different eukaryotic expression vectors, the T295/G350 mutation site promoted the proliferation, migration, and invasion of C33A cells to a greater extent than G295/G350; however, G295/G350 had a stronger effect than T295/T350.

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.

Low Frequency of Human Cytomegalovirus in Cancerous and Precancerous Cervical Samples of Iranian Women

Background: HPV-16 has a significant role in cervical cancers; co-infection with human cytomegalovirus (HCMV) as an oncomodulatory pathogen may increase the risk of carcinogenesis. This study aimed to investigate the frequencies of HCMV and HPV-16 in cervical samples. Materials &amp; methods: A total of 102 cancerous and precancerous cervical samples were examined by real-time PCR targeting the HPV-16 E6 gene, and HCMV immediate-early gene. Results: In total, 65 samples (63.7%) were positive for HPV-16. HCMV was found in seven samples (6.9%). Both HPV-16 and HCMV were present in four samples (cervical intraepithelial neoplasia-3 and squamous cell carcinoma groups with two samples each). Conclusion: HCMV can infect cervical tissues at a low frequency, suggesting that HCMV is unlikely to play a role in the cervical carcinogenesis.

Immunohistochemical Detection of Human Papillomavirus 16 E7 Oncoprotein in Cervical Lesions.

Almost all cervical cancer is associated with persistent infection of high-risk (hr) human papillomavirus (HPV) like HPV16. The E7 oncoprotein encoded by hrHPV plays a crucial role in carcinogenesis. The present study aimed to establish a reliable protocol of immunohistochemistry stains to detect HPV16 E7 protein in formalin-fixed and paraffin-embedded tissue specimens of various cervical lesions. Firstly, the HPV16 E7 gene was inserted into a prokaryotic expression vector pGEX-4T2. Then the recombinant plasmid pGEX-4T2-(HPV16-E7) was transformed into Escherichia coli JM109. The fusion protein containing a GST tag was purified, and New Zealand white rabbits were immunized to produce the HPV16 E7 polyclonal antibody. The anti-HPV16 E7 antibody was verified by western blotting and immunofluorescence stains, and applied in 182 HPV16 DNA-positive cervical tissue specimens and matched 36 HPV DNA-negative specimens by immunohistochemistry. Furthermore, A positive correlation between HPV16 E7 protein expression and malignancy grade was observed. But there is no relationship between HPV16 E7 protein expression and tumor sizes, tumor differentiation, lymph node metastasis, International Federation of Gynecology and Obstetrics (FIGO) stage, or lymphovascular space invasion in cervical cancer. These findings provide a basis for further research focusing on the role of HPV E7 protein in various HPV-related diseases.

HPV16 E6 regulates the proliferation, invasion, and apoptosis of cervical cancer cells by downregulating miR-504.

BackgroundHuman papillomavirus (HPV) 16 infection is a necessary condition for the pathogenesis and development of cervical cancer. The E6 protein is expressed by the HPV16 E6 gene and promotes malignant phenotype transformation, which is an important mechanism for the occurrence and development of cervical cancer. MicroRNA-504 (miR-504) has been reported as an oncogene or tumor suppressor gene; the expression of miR-504 in cervical cancer has been found to be negatively correlated with HPV infection. However, the relationship between HPV16 E6 and miR-504 and the role of miR-504 in cervical cancer are not clear. In the current study, we observed the effect of HPV16 E6 on the expression of miR-504 in cervical cancer cells, and analyzed whether HPV16 E6 affects proliferation, invasion, and apoptosis in cervical cancer cells by regulating the expression of miR-504.MethodsCervical cancer cells (SiHa) were divided into four groups: the empty vector group, E6 overexpression group, E6 overexpression + miR-NC group, and E6 overexpression+miR-504 group. The expressions levels of HPV16 E6 mRNA and miR-504 were detected by real-time polymerase chain reaction (PCR), and the expression level of HPV16 E6 protein was detected by Western blot. Cell proliferation, invasion, and apoptosis were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Tastelessly, and flow cytometry, respectively.ResultsThe expression level of miR-504 was significantly decreased in E6 overexpression cells compared to the control cells (P<0.05); the overexpression of miR-504 with miR-504 mimic significantly reversed the downregulation of miR-504 in E6 overexpression SiHa cells (P<0.05). MTT and Transwell assays showed that the overexpression of E6 significantly increased proliferation and invasion of SiHa cells (P<0.05). The overexpression of miR-504 reversed the role of HPV16 E6 on the proliferation and invasion in E6 overexpression SiHa cells, and the difference was statistically significant (P<0.05). Further analysis showed that the overexpression of E6 significantly reduced apoptosis of SiHa cells (P<0.05). The overexpression of miR-504 reversed the role of HPV16 E6 on apoptosis in E6 overexpression SiHa cells, and the difference was statistically significant (P<0.05).ConclusionsHPV16 E6 may promote the proliferation and invasion, and inhibit the apoptosis, of cervical cancer SiHa cells by downregulating miR-504 expression.

PEG-PtS2 nanosheet-based fluorescence biosensor for label-free human papillomavirus genotyping.

Asimple and efficient ultrasonication-assisted liquid exfoliation method is proposedto produce PtS2 nanosheets on a large scale and improve their dispersion in aqueous solution by surface polyethylene glycol modification. Theinteraction of polyethylene glycol-modified PtS2 (PEG-PtS2) nanosheets with fluorescent labeled DNA and the fluorescence quenching mechanism using FAM-labeled hpv16e6 gene fragment as aprobewas investigated. The excitation and emission wavelengths were 468 and 517nm, respectively. The fluorescence quenching mechanism of PEG-PtS2 nanosheets for double-stranded DNA (dsDNA) might stem from thestatic quenching effect. Based on the difference in fluorescence quenching capability of PEG-PtS2 nanosheets in fluorescent probe tagged single-stranded DNA (ssDNA) and dsDNA, a mix-and-detect method was proposed for determination of DNA. Without the need for probe immobilization and tedious washing steps, the genotyping of human papillomavirus (HPV) was easily achieved. The limit of detection was calculated to0.44nM, showing a good linear range within 0.05-10nM. We believe this biosensor provides opportunities to develop asimple and low-cost strategy for molecular diagnostics. Graphical abstract.

Association between APOBEC3s and HPV16 E2 gene hypermutation in Uygur females with cervical cancer.

The present study aimed to investigate whether apolipoprotein B mRNA-editing enzyme catalytic polypeptides (A3) are involved in the regulation of cervical cancer development and human papilloma virus (HPV)16 sustained infection in Uighur females. Cervical tissues of Uygur patients with HPV16 with cervical lesions were collected. Expression levels of A3C, A3F and A3G were detected using reverse transcription-quantitative PCR and western blotting. A model of SiHa cells with high expression levels of A3C, A3F and A3G was constructed. Hypermutation was detected using the differential DNA denaturation PCR and positive samples were amplified and sequenced. There were significant differences in A3 expression levels in cervical lesions of different grades. A3C and A3F mRNA and protein expression in cervical cancer tissues were significantly lower, whereas the A3G mRNA and protein expression levels were significantly higher compared with the cervicitis and cervical intraepithelial neoplasia (CIN) I–III groups. Hypermutation rates were increased with cervical lesion development. C>T and G>A base substitutions were detected in all hypermutation samples and numbers of C>T and G>A base substitutions in single samples in the cervical cancer group were significantly higher compared with those in the CIN I–III and cervicitis groups. Following transfection of A3F and A3G, HPV E2 mRNA and protein expression levels were significantly decreased in SiHa cells. Numerous C>T and G>A base substitutions were detected in the HPV E2 gene in A3G and A3C overexpressing SiHa cells. A3 family proteins inhibit viral replication during HPV16 infection and regulate the HPV16 integration by inducing C>T and G>A hypermutations in the HPV16 E2 gene, thus affecting the cervical cancer pathogenesis and development.

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.

The expression of the transcription factor TAF1 is modified by the HPV16 E2 protein.

High-risk human papillomaviruses (e.g., HPV16 and 18) are associated with cervical cancer occurrence and development. The early viral gene E2 encodes a protein involved in several key processes in HPV biology, such as replication, genome segregation, and viral gene transcription. E2's presence also affects the expression of a variety of cellular genes involved in a wide range of biological processes, including cell cycle regulation and apoptosis, which are mediated by E2's interaction with cellular proteins. In this report, a lentiviral system was used to express the HPV16 E2 gene in the HPV-negative C-33A cell line for several weeks. E2 expression was measured by RT-qPCR and its biological activity was evaluated using a reporter gene. In HPV16 E2-positive cells, we observed a statistically significant increase in mRNA and protein levels of TAF1 and p27, a basal transcription factor and one of its target genes, respectively. To our knowledge, this is the first study showing that the viral protein HPV16 E2 upregulates TAF1 expression. This suggests that E2's expression promotes a transcriptionally-favorable cellular environment that allows HPV to successfully complete its replication cycle. Keywords: HPV16; E2 protein; transcription; TAF1 regulation.

Targeting interferon signaling and CTLA-4 enhance the therapeutic efficacy of anti-PD-1 immunotherapy in preclinical model of HPV+ oral cancer

BackgroundThe US is experiencing an epidemic of HPV+ oropharyngeal cancers (OPC), the rates and burden of which now exceed that for cervical cancer. Immunotherapy targeting programmed death 1 (PD-1) on tumor-infiltrating lymphocytes and/or its ligand PD-L1 on tumor cells, which was effective in several cancers has however, showed efficacy in only less than 15% of patients.MethodsWe used a preclinical HPV+ oral tumor model, mEER, consisting of mouse tonsil derived epithelial cells expressing HPV-16 E6 and E7 genes, along with the H-ras oncogene to test strategies for enhancing the efficacy of anti-PD-1 therapy.ResultsMonotherapy with PD-1 blocking antibody was ineffective against flank-implanted tumors, but induced regression in 54% of mice bearing orthotopic tongue tumors that correlated with higher CD8 T cell responses. Since the CD8+ T cells derived from tongue tumors also showed high levels of the immune checkpoint inhibitory receptor CTLA-4, we tested combination immunotherapy targeting both CTLA-4 and PD-1 together and observed 93.3% survival of mice bearing tumors in the tongue for the duration of our 100-day study. Protective immunity correlated with a significant decrease in immunosuppressive lymphoid and myeloid populations within the tumor microenvironment. Consistent with the reported capacity of interferon-driven PD-L1/PD-1 pathway induction to serve as a biomarker of response to PD-1 blockade, we observed elevated interferon signaling and significantly higher levels of PD-1/PD-L1 in tongue-implanted mEER tumors compared to those growing on the flank correlating with their preferential responsiveness to PD-1 blockade. More importantly, in a pseudometastasic mouse model bearing both flank and tongue tumors to represent metastatic disease, delivery of Stimulator of Interferon Induced Genes (STING) agonist into the flank tumors combined with systemic treatment with α-PD-1 and α-CTLA-4 antibodies resulted in sustained tumor regression in 71% of mice. In this case, productive abscopal anti-tumor immunity was associated with robust increases in the ratios of cytotoxic CD8+ T cells (CTL) versus regulatory T cells (Treg) and versus functional myeloid-derived suppressor cells (MDSC).ConclusionsThese results support combining α-PD-1 therapy with induction of IFN-α/β signaling via provision of STING agonist and/or through CTLA-4 blockade as potential treatment option for HNSCC patients, especially, those not responding to α-PD-1 monotherapy.

Human papilloma virus DNA-biomarker analysis for cervical cancer: Signal enhancement by gold nanoparticle-coupled tetravalent streptavidin-biotin strategy

Human papillomavirus (HPV) is a double-standard DNA virus, as well as the source of infection to the mucous membrane. It is a sexually transmitted disease that brings the changes in the cervix cells. Oncogenes, E6 and E7 play a pivotal role in the HPV infection. Identifying these genes to detect HPV strains, especially a prevalent HPV16 strain, will bring a great impact. Among different sensing strategies for pathogens, the dielectric electrochemical biosensor shows the potential due to its higher sensitivity. In this research, HPV16-E7 DNA sequence was detected on the carbodiimidazole-modified interdigitated electrode (IDE) surface with the detection limit of 1 fM. To enhance the sensitivity, the target sequence was conjugated on gold nanoparticle (GNP) and attained detection to the level of 10 aM. This produced ~100 folds improvement in detecting HPV16-E7 gene and 4 folds increment in the current flow. The stability of HPV16-E7 DNA sequences on GNP was verified by the salt-induced GNP aggregation. The current system has shown the higher specificity by comparing against non-complementary and triple-mismatched DNA sequences of HPV16-E7. This demonstration in detecting HPV16-E7 using dielectric IDE sensing system with a higher sensitivity can be recommended for detecting a wide range of disease-causing DNA-markers.