HPV Gene Research Articles

Abstract 4621: Risk stratification and biomarker discovery in HPV-positive oropharynx squamous cell carcinoma determined by HPV and human gene expression profile associations

Abstract Determining which HPV+ oropharyngeal squamous cell carcinoma (OPSCC) patients will respond to therapy is of utmost important for implementation of treatment de-escalation to reduce morbidity or testing of novel therapeutic approaches. Due to the lack of reliable indicators of treatment response, we explored the gene expression profile of OPSCC in order to better understand the biology of these tumors and identify novel biomarkers. In this study, we investigated human genes associated with HPV transcription in 80 OPSCC samples from The Cancer Genome Atlas (TCGA). This exploratory analysis provided a list of 582 human genes associated with HPV biology. Hierarchical clustering analysis using the 582 human genes was used to separate HPV+ OPSCC in two groups with significantly distinct survival (log-rank test p < 0.001) and differential expression of HPV genes. A refinement of this analysis generated a prognostic gene expression signature for HPV+ OPSCC containing 41 human genes. These results were validated in two independent cohorts of HPV+ OPSCC (n=83 and n=47) and one independent cohort of cervical cancer (n=83). In all three validation cohorts our 41 gene expression signature was able to identify a group of HPV+ OPSCC patients with worse survival. Further refinement and validation of the signature is ongoing. In order to understand the biology related to poor outcome in HPV+ OPSCC, a whole transcriptome analysis between the two initial HPV+ OPSCC TCGA groups was performed. Pathway analysis identified cell metabolism, cell stress, and DNA damage related genes were highly associated with poor outcome. Similar patterns of gene expression were found in vitro in a panel of 10 HPV+ cell lines, and markers of the poor outcome were found to be associated with reduced radiation sensitivity in vitro. Our study has identified prognostic biomarkers in HPV+ OPSCC with potential clinical importance. These biomarkers may be useful for selection of low risk patients for treatment de-escalation or selection of high risk patients for novel therapeutic approaches. These biomarkers are also functionally linked to radiation sensitivity and may include new therapeutic targets. Citation Format: Frederico O. Gleber-Netto, Xiayu Rao, Kelly Erikson, Keiko Akagi, Faye M. Johnson, Jing Wang, Joseph Califano, Maura L. Gillison, Jeffrey N. Myers, Curtis R. Pickering. Risk stratification and biomarker discovery in HPV-positive oropharynx squamous cell carcinoma determined by HPV and human gene expression profile associations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4621.

Adenosine causes read-through into the late region of the HPV16 genome in a guanosine-dependent manner

Adenosine plays an important role in cell death and differentiation as well as in tumorigenesis and the intra- and extra-cellular levels range from nanomolar to millimolar levels under various physiological or pathophysiological conditions. Here we report that adenosine can activate HPV16 late gene expression in a dose- and time-dependent manner, but only in the presence of guanosine. This activation occurred within hours after addition of the nucleosides and was primarily dependent on the ENT1 nucleoside transporter protein. Induction of HPV16 late gene expression was mainly the result of increased read-through at the early HPV16 polyadenylation signal into the late region of the HPV16 genome, thereby producing HPV16 late L2 mRNAs. The effect of guanosine and adenosine on HPV16 late gene expression was mediated by the increased binding to HPV16 mRNAs and nuclear export of the cellular HuR protein. Our results demonstrate that nucleosides can affect HPV16 gene expression.

Induction of Interferon Kappa in Human Papillomavirus 16 Infection by Transforming Growth Factor Beta-Induced Promoter Demethylation.

Persistent high-risk human papillomavirus (HPV) infection is the major causal factor in cervical and other anogenital cancers. Because there are currently no therapeutics capable of preventing neoplastic progression of HPV infections, understanding the mechanisms of HPV-mediated persistence, including immune evasion, is a major research priority. The multifunctional growth factor transforming growth factor beta (TGFβ) has been shown to inhibit expression of early viral transcripts from cells harboring integrated HPV genomes or cells infected with retroviruses expressing HPV oncoproteins. However, the mechanism of TGFβ-induced inhibition has not been fully defined. In this study, we have observed a previously uncharacterized ability of TGFβ to repress the differentiation-induced upregulation of late HPV16 gene expression. In addition, interferon kappa (IFN-κ), a keratinocyte-specific, constitutively expressed cytokine suppressed by differentiation, can be transcriptionally induced by TGFβ1. TGFβ-mediated IFN-κ transcription only occurs in cells containing HPV16, and this is due to TGFβ1-mediated reversal of HPV-induced methylation of the IFN-κ promoter through active DNA demethylation mediated by thymine DNA glycosylase (TDG). This novel interaction between growth factor and innate immune signaling may shed light on the mechanisms of HPV persistence and how the virus manipulates both immune and growth factor signaling to promote its life cycle.IMPORTANCE Persistent infection by high-risk HPVs is the primary risk factor for development of HPV-induced cancers. Persistence involves viral evasion of the immune response, including the IFN response. HPV is also known to suppress TGFβ signaling, which inhibits viral gene expression. Here, we show that the TGFβ and IFN pathways are interrelated in the context of HPV16 infection through the upregulation of IFN-κ by TGFβ. The ability of TGFβ to induce IFN-κ promoter demethylation and transcriptional activation provides a new explanation for why HPV has evolved mechanisms to inhibit TGFβ in infected cells.

Demethylation Therapy as a Targeted Treatment for Human Papillomavirus-Associated Head and Neck Cancer.

Purpose: DNA methylation in human papillomavirus-associated (HPV+) head and neck squamous cell carcinoma (HNSCC) may have importance for continuous expression of HPV oncogenes, tumor cell proliferation, and survival. Here, we determined activity of a global DNA-demethylating agent, 5-azacytidine (5-aza), against HPV+ HNSCC in preclinical models and explored it as a targeted therapy in a window trial enrolling patients with HPV+ HNSCC.Experimental Design: Sensitivity of HNSCC cells to 5-aza treatment was determined, and then 5-aza activity was tested in vivo using xenografted tumors in a mouse model. Finally, tumor samples from patients enrolled in a window clinical trial were analyzed to identify activity of 5-aza therapy in patients with HPV+ HNSCC.Results: Clinical trial and experimental data show that 5-aza induced growth inhibition and cell death in HPV+ HNSCC. 5-aza reduced expression of HPV genes, stabilized p53, and induced p53-dependent apoptosis in HNSCC cells and tumors. 5-aza repressed expression and activity of matrix metalloproteinases (MMP) in HPV+ HNSCC, activated IFN response in some HPV+ head and neck cancer cells, and inhibited the ability of HPV+ xenografted tumors to invade mouse blood vessels.Conclusions: 5-aza may provide effective therapy for HPV-associated HNSCC as an alternative or complement to standard cytotoxic therapy. Clin Cancer Res; 23(23); 7276-87. ©2017 AACR.

Maintenance of HPV Gene Expression and Decreased Local Immune Response During Chemoradiation are Associated With Death From Cervical Cancer

Maintenance of HPV Gene Expression and Decreased Local Immune Response During Chemoradiation are Associated With Death From Cervical Cancer

HnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner.

Inhibition of the Akt kinase activates HPV16 late gene expression by reducing HPV16 early polyadenylation and by activating HPV16 late L1 mRNA splicing. We identified ‘hot spots’ for RNA binding proteins at the early polyA signal and at splice sites on HPV16 late mRNAs. We observed that hnRNP L was associated with sequences at all HPV16 late splice sites and at the early polyA signal. Akt kinase inhibition resulted in hnRNP L dephosphorylation and reduced association of hnRNP L with HPV16 mRNAs. This was accompanied by an increased binding of U2AF65 and Sam68 to HPV16 mRNAs. Furthermore, siRNA knock-down of hnRNP L or Akt induced HPV16 gene expression. Treatment of HPV16 immortalized keratinocytes with Akt kinase inhibitor reduced hnRNP L binding to HPV16 mRNAs and induced HPV16 L1 mRNA production. Finally, deletion of the hnRNP L binding sites in HPV16 subgenomic expression plasmids resulted in activation of HPV16 late gene expression. In conclusion, the Akt kinase inhibits HPV16 late gene expression at the level of RNA processing by controlling the RNA-binding protein hnRNP L. We speculate that Akt kinase activity upholds an intracellular milieu that favours HPV16 early gene expression and suppresses HPV16 late gene expression.

Abstract 4794: Reduced Epstein-Barr virus replication in human papillomavirus-immortalized keratinocyte organotypic raft culture

Abstract An epidemic rise in oropharyngeal squamous cell carcinoma (OSCC) infected with human papillomavirus has been observed over the past decade. The majority of HPV+OSCC arise from the tonsils and base of tongue (BOT), which are lymphoid-rich regions that are known to harbor Epstein-Barr virus (EBV). Indeed, we previously reported HPV/EBV co-infection in 25% of tonsillar and 70% of BOT tumors. HPV is a DNA tumor virus that specifically infects proliferating basal cells in stratified squamous epithelia. HPV replication is facilitated by viral oncoproteins E6 and E7, which create an S-phase-like environment via degradation of cell cycle regulators p53 and Rb, respectively. EBV is also an established DNA tumor virus that replicates in differentiated epithelia in its normal lifecycle. However, EBV exhibits a latent infection characterized by oncogene expression and lack of viral replication in EBV-associated carcinomas. E6/E7-mediated alteration of the cell cycle results in an interruption in epithelial differentiation, which is required for EBV replication in epithelial cells. Thus, we hypothesize that HPV immortalization of keratinocytes shifts the EBV life cycle from replication to latency. To test our hypothesis, we developed an EBV/HPV co-infection model utilizing organotypic raft culture to produce stratified epithelia in vitro. Tonsillar keratinocytes immortalized by HPV were EBV-infected by co-culture with IgG-induced EBV-positive Akata Burkitt’s lymphoma cells four days after differentiation induction. Raft tissues were harvested 6 days post-EBV infection for analysis of viral life cycles. EBV infection did not alter HPV replication or gene expression. In HPV-negative tonsillar keratinocytes, robust EBV replication was observed that was blocked by treatment with acyclovir, a nucleoside analog that inhibits EBV lytic replication. In contrast, a dramatic decrease in EBV DNA levels was observed in HPV-positive keratinocytes, which was not further reduced by acyclovir. Human telomerase-immortalized normal oral keratinocytes supported robust EBV replication in organotypic raft culture suggesting a specific effect of HPV rather than immortalization on EBV replication. Expression of E7 alone was sufficient to decrease EBV DNA levels. Preliminary data suggest no change in EBV infectivity between HPV-negative and HPV-positive rafts, and an increase in EBV latency-associated gene expression in HPV-positive cells. The ability of EBV to establish persistent latent infection in HPV-infected epithelial cells would contribute additional viral oncogene expression to explain the rapid development and progression of HPV+OSCC. Citation Format: Joseph T. Guidry, William K. Songock, Xiaohui Ma, Cherie-Ann O. Nathan, Jason M. Bodily, Rona S. Scott. Reduced Epstein-Barr virus replication in human papillomavirus-immortalized keratinocyte organotypic raft culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4794. doi:10.1158/1538-7445.AM2017-4794

Abstract 4800: A next-gen animal model to Study PIK3CA-mTOR driven HPV-related oral malignancies

Abstract The rising incidence of HPV-associated malignancies, especially for head and neck squamous cell carcinoma (HNSCC), has highlighted the urgent need to understand the role of HPV-16 E6/E7 oncogenes in cancer initiation and progression. For that propose we decided to generate a doxycycline inducible E6/E7 transgenic mouse targeting E6/E7 onco-proteins to the basal epithelia layer, including the stem cell compartment, through a doxycycline inducible cytokeratin 5 promoter (cK5-rtTA). This system allows to turn on the expression of HPV genes after birth, minimizing the negative effect that viral E6/E7 expression may have during embryonic development, and mimicking the natural infection process that usually happens in adults after initiation of sexual activities. Interestingly, we found that after doxycycline induction, both E6 and E7 were highly expressed, resulting in a clear increase in the number of proliferative cells even in the suprabasal layers, which results in rapid epidermal hyperplasia. However, in our animal mouse model HPV-16 E6/E7 expression alone was not sufficient to induce spontaneous squamous carcinomas (SCC). Due that we decided to challenge the oncogenic role of HPV using a classical chemical carcinogenesis protocol (DMBA-TPA). These mice develop SCC rapidly after a single exposure to a skin carcinogen, DMBA, which was increased by the prolonged exposure to a tumor promoter, TPA. These data suggest that only few oncogenic hits may be sufficient to induce cancer in humans already infected with HPV-16 E6/E7. Then, we decided to elucidate whether or not a few genomic alterations in combination with HPV-16 expression may be sufficient to induce cancer. One of the most frequent mutations in HNSCC, including HPV-associated cancers, are those affecting the PI3K-mTOR pathway. Based on that, we engineered a genetically defined mouse model combining HPV-16 E6/E7 and PIk3CAH1047R expression under the control of a cytokeratin 14 promoter driven tamoxifen-regulated Cre recombinase (cK14-CreERTM). Surprisingly, we found that upon tamoxifen activation mice develop oral cancer in only few weeks. Furthermore, our HPV-related cancer mouse models exhibited a marker increase of mTOR activation which is a key oncogenic driver in malignant progression to SCC. Remarkably, rapamycin, an mTOR inhibitor, was able to block tumor development, supporting the potential clinical use of direct and indirect mTOR inhibitors as a molecular targeted approach for prevention of HPV-associated malignancies. Citation Format: Juan Luis Callejas-Valera, Ramiro Iglesias-Bartolome, Panomwat Amornphimoltham, Julia Palacios-Garcia, Daniel Martin, Joseph A. Califano, Alfredo A. Molinolo, J.Silvio Gutkind. A next-gen animal model to Study PIK3CA-mTOR driven HPV-related oral malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4800. doi:10.1158/1538-7445.AM2017-4800

Selective p300 inhibitor C646 inhibited HPV E6-E7 genes, altered glucose metabolism and induced apoptosis in cervical cancer cells

High risk HPV infection is a causative factor of cervical cancer. The constitutive expression of HPV E6-E7 genes is important for the maintenance of cancer phenotypes. The cellular transcription co-activator p300 plays a crucial role in the regulation of HPV genes thus it was targeted for the inhibition of HPV-associated cervical cancer. In the present study, HPV positive cervical cells were treated with C646, a selective inhibitor of p300, to investigate its influence on HPV E6-E7 expression and cancer cell growth. Results of RT-qPCR, Western-blot and promoter activity assays showed that C646 inhibited the transcription of HPV E6-E7, which was accompanied with the accumulation of p53 protein. Meanwhile, cell proliferation was suppressed, glucose metabolism was disrupted and apoptosis was induced via the intrinsic pathway. Generally, the anti-cervical cancer potential of C646 was demonstrated and a novel mechanism was proposed in this study.

HPV positive neuroendocrine cervical cancer cells are dependent on Myc but not E6/E7 viral oncogenes

Using conditional cell reprogramming, we generated a stable cell culture of an extremely rare and aggressive neuroendocrine cervical cancer. The cultured cells contained HPV-16, formed colonies in soft agar and rapidly produced tumors in immunodeficient mice. The HPV-16 genome was integrated adjacent to the Myc gene, both of which were amplified 40-fold. Analysis of RNA transcripts detected fusion of the HPV/Myc genes, arising from apparent microhomologous recombination. Spectral karyotyping (SKY) and fluorescent-in-situ hybridization (FISH) demonstrated coordinate localization and translocation of the amplified Myc and HPV genes on chromosomes 8 and 21. Similar to the primary tumor, tumor cell cultures expressed very high levels of the Myc protein and, in contrast to all other HPV-positive cervical cancer cell lines, they harbored a gain-of-function mutation in p53 (R273C). Unexpectedly, viral oncogene knockdown had no effect on the growth of the cells, but it did inhibit the proliferation of a conventional HPV-16 positive cervical cancer cell line. Knockdown of Myc, but not the mutant p53, significantly inhibited tumor cell proliferation. On the basis of these data, we propose that the primary driver of transformation in this aggressive cervical cancer is not HPV oncogene expression but rather the overexpression of Myc.

Heat-Induced Editing of HPV Genes to Clear Mucocutaneous Warts?

Hyperthermia increases expression of the antiviral cellular factors APOBEC3A and APOBEC3G and induces G-to-A or C-to-T mutations in human papilloma virus cervical cell lines and genital warts. This unexpected effect of heat treatment correlated with regression of genital warts in a subset of patients, including at distant sites, suggesting that this effect may be mediated in part by antiviral as well as immunological mechanisms.

Abstract A27: A linked data approach to discover HPV oncoprotiens and RB1 induced mutation associations for the retinoblastoma research

Abstract Background: LOSS or GAIN in tumor suppressor gene RB1 play a significant role as in case of loss low penetrance where only 39% of eye at risk develops in retinoblastoma. This research covers the multiple mutation types and its effects and identification of major type of mutation involved in retinoblastoma because of HPV and RB1. Methods: First, we focus on exploring gene expression (GE) patterns for RB1 and HPV associated genes from TCGA. Second, identification of validated and non-validated standard CNV ensured using the COSMIC. Finally, the clinical profiles of filtered mutations have been validated based on ICGC pathological profiling data to infer the prognostic behavior from RB1 and HPV associated genes. In order to link and retrieve patterns of a gene from TCGA, COSMIC, and ICGC repositories, we performed following steps: transform heterogeneous data repositories and their storage formats into standard Resource Description Framework (RDF) format; to discover associations by finding specific patterns (i.e. correlations) in the GE data sets; scalable querying the large volume and frequently updating datasets covering the GE data from different repositories. Results: HPV mutations indicated in more than 127 cancer studies shows deletion and amplifications are rare mutations. Retinoblastoma: Expression profile of RB1 shows mutations such as nonsense, Missense or splice events and in GBM and gliomas the expression values in splice mutations (1500-200), nonsense mutations (200-600). In principal HPV associated retinoblastoma the higher expression of HPV genes results in splice junctions and lower in nonsense mutations. Other tissues: Pattern of RB1 where the results coming from more 123 studies show the pattern of mutations similar to the results obtained from HPV associated genes. Alteration with HPV genes study based on the alteration in Altered in 90% samples of 61 cases where TP53 is holding 90% occurrence majorly as normal mutations and SNRNP70 and BRCA1 is majorly responsible truncating mutation other highly mutated genes are AP3D1, BRD4, CCHCR1, CPSF4, CREBBP, CUL3, DDX11, EP300, EP400, FRZ1, GNB2L1, GTF2B, KDM5C, NR4A1, PRP1, PLK1, SF1, SRSF1, SRSF7, SMARCB1, SNRNP70, TAF1, TBP, TMF1, TOPBP1. Whereas RB1 is associated with 11% cased of deep deletion where the V654L is the normal mutation and all others are highly truncating mutation. Survival graph for HPV and RB1 associated genes median months(m) of survival with alteration in these query genes are 103m whereas in RB1 the median month of survival are 7.63m however the disease free survival in RB1 cases are 4.50m. The p-value are 0.76, 0.67, 0.38 respectively. To demonstrate pattern of survival gene set enrichment have been performed on both gene lists where in case of RB1 genes with highest interactions are MDM2, CDK4 and TP53. In HPV genes interacting hubs are TOP1, PARP1, TP53 and ODF2. Higher interacting genes are associated with drugs. RB1 corresponded to Insulin, p a non-cancer FDA approved drug whereas HPV genes and especially TOP1 is associated with Lucanthone, Innotecan, BTBD1 and Topotecan in case of FDA approved drugs category. Cancer drugs with HPV genes are majorly associated with TOP1, PARP1 and PLK1 namely BTBD1, AZD 2281, AG14361, BI2536 and GW84382X. In ICGC effect of RB1 is on cancers e.g. melanoma 51.91% Esophageal 45.38% ovarian 31.18% liver 27.96% Pancreatic 22.55%. Associations of RB1 with other cancer mutations are either splice as in TCGA and other associated mutation with RB1 is LPAR6 majorly these mutations are in exon-region and further understand the other mutations in TCGA ICGC reveals most of these are SNPs at chromosome-13 which defines the locus of RB1 and for HPV. Higher interacting hubs from TCGA TOP1, PAPR1 and ODF2. TOP1 is associated with melanoma two donor hubs of 42.08% and 40.91% liver cancer Hepatocellular carcinoma (Virus) with 23.08 % Esophageal (15.05%) and Ovarian (11.36%). Mutations types are SNP and Splice junctions. Citation Format: Alokkumar Jha, Yasar Khan, Dietrich Rebholz-Schumann, Ratnesh Sahay. A linked data approach to discover HPV oncoprotiens and RB1 induced mutation associations for the retinoblastoma research. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A27.

Interstitial collagenase and their endogenous regulators in squamous cell cervical carcinoma

Interstitial collagenase (MMP-1) belongs to the family of matrix metalloproteinases (MMP), which play a key role in generalization processes of invasion and metastasis, which determine the degree of tumor malignancy. MMP-1 refers to secreted, inducible MMP, the expression of which in normal tissues is not defined. Induction of expression of MMP in CSS in the tumor occurs under the action of oncogenes of HPV, and in areas adjacent to the tumor normal tissue under the action of the inductor expression of MMP - EMMPRIN (CD147), which expressively on the surface of tumor cells. The aim of this study is to determine the possibility of expression ofMMP-1 and its regulators (tissue inhibitors TIMP-1 and activator - plasminogen activator - ADF) in morphologically normal body of the uterus during CSS. The study was carried out using on a tissue "tape" - postoperative specimens of the uterus when the diagnosis of CSS. All of the samples was expressed HPV16 gene E7. It was shown that: 1. The increase of MMP-1 expression, low expression (or lack thereof) of its inhibitors TIMP-1 and a very clear expression of the activator take place in the tumor when CSS that lead to increased activity of MMP-1, and aims to increase the destructive (invasive) potential of the tumor. 2. In morphologically normal tissue of the uterus during CSS the expression of MMP-1 can occur from the vaginal wall to the bottom of the uterine cavity, but at a much lower level than in the tumor. 3. These data indicate the possibility of development of a destructive process in morphologically normalnyh body tissues of the uterus during CSS, important for understanding the mechanism of tumor progression, and suggest participation in the process of expression of MMP-1 signaling by the type of epithelial-mesenchymal interaction .

Association between human papillomavirus (HPV) 16, HPV18, and other HR-HPV viral load and the histological classification of cervical lesions: Results from a large-scale cross-sectional study.

The relationship between HPV viral load and histological grades in the development of cervical cancer is in argument. It is helpful to better understand the association by quantitatively detecting viral load of HPV16, 18, and a pool of 12 other high-risk HPV type (OT) independently on the samples of precancer and cancer. A cross-sectional study was performed in five medical centers of China. Histological diagnosis made by local pathologists was adjudicated via a pathology expert panel. A fully automated real-time PCR test was used for the measurement of HPV16, 18, OT, and human β-globin gene. A total of 2,513 women [1,341 normal, 209 low grade intraepithelial lesion (LSIL), 392 high grade intraepithelial lesion (HSIL), 520 squamous cell carcinoma (SCC), and 51 adenocarcinoma (ADC)] were included. There is a linear increase in the total 14 HPV viral load with histological grade from normal to SCC. This trend was not observed in HPV18 infection but HPV16. The viral load for OT was low in normal, peaked in LSIL and HSIL, and declined in SCC and ADC. In the co-infection of HPV16 and HPV18, HPV16 viral load was significantly higher than HPV18 in LSIL and HSIL. In co-infection of HPV16 and OT, higher HPV16 viral load was also seen in SCC and ADC. Viral load of HPV16 increases with cervical lesion grade and is predominant in cervical cancer. HPV18 viral load is low in precancer, but going up in cancer. OT viral load shows inverse trend of HPV18. J. Med. Virol. 89:535-541, 2017. © 2016 Wiley Periodicals, Inc.

Higher prevalence and gene amplification of HPV16 in oropharynx as compared to oral cavity.

ABSTRACTObjective The objective of this study was to clarify differences regarding HPV16 infection and gene amplification between the oral cavity and oropharynx in healthy individuals.Material and Methods The subjects were 94 healthy asymptomatic individuals (41 males, 53 females; mean age 58.6 years, range 16-97 years) who visited the Department of Oral and Maxillofacial Reconstructive Surgery of the Hiroshima University Hospital from 2014 to 2015. Oral epithelial cells were collected from oral rinse and pharynx gargle samples and placed in saline. The human endogenous retrovirus gene ERV3-1 was used as a reference to estimate the number of human cells in each sample. DNA samples were extracted from approximately 10,000 human cells and tested for HPV16 DNA by PCR using a type-specific primer. Similarly, we analyzed the HPV16 viral copy number in HPV16-positive cases using real-time PCR to examine genomic amplification.Results The percentage of HPV16-positive cases was higher in the gargle (28.7%) as compared to the rinse (16.0%) samples. In the oral rinse samples, males (26.8%) showed a significantly higher rate of HPV16 than females (7.5%) (P=0.021). Importantly, in older subjects (aged 60-89 years), gargle samples showed a significantly higher rate of HPV16 (33.3%) than oral rinse samples (13.7%) (P=0.034). The average number of viral copies was approximately 8 times higher in the gargle than in the oral rinse samples (0.16±0.27 vs. 1.35±1.26 copy numbers per cell), a significant difference (P<0.001).Conclusion Our findings suggest that the oropharynx is more susceptible to HPV16 infection as compared to the oral cavity, while HPV16 gene amplification is also more commonly found in the oropharynx.

Production of functional, stable, unmutated recombinant human papillomavirus E6 oncoprotein: implications for HPV-tumor diagnosis and therapy.

BackgroundHigh-risk human papillomaviruses (HR-HPVs) types 16 and 18 are the main etiological agents of cervical cancer, with more than 550,000 new cases each year worldwide. HPVs are also associated with other ano-genital and head-and-neck tumors. The HR-HPV E6 and E7 oncoproteins are responsible for onset and maintenance of the cell transformation state, and they represent appropriate targets for development of diagnostic and therapeutic tools.MethodsThe unmutated E6 gene from HPV16 and HPV18 and from low-risk HPV11 was cloned in a prokaryotic expression vector for expression of the Histidine-tagged E6 protein (His6-E6), according to a novel procedure. The structural properties were determined using circular dichroism and fluorescence spectroscopy. His6-E6 oncoprotein immunogenicity was assessed in a mouse model, and its functionality was determined using in vitro GST pull-down and protein degradation assays.ResultsThe His6-tagged E6 proteins from HPV16, HPV18, and HPV11 E6 genes, without any further modification in the amino-acid sequence, were produced in bacteria as soluble and stable molecules. Structural analyses of HPV16 His6-E6 suggests that it maintains correct folding and conformational properties. C57BL/6 mice immunized with HPV16 His6-E6 developed significant humoral immune responses. The E6 proteins from HPV16, HPV18, and HPV11 were purified according to a new procedure, and investigated for protein–protein interactions. HR-HPV His6-E6 bound p53, the PDZ1 motif from MAGI-1 proteins, the human discs large tumor suppressor, and the human ubiquitin ligase E6-associated protein, thus suggesting that it is biologically active. The purified HR-HPV E6 proteins also targeted the MAGI-3 and p53 proteins for degradation.ConclusionsThis new procedure generates a stable, unmutated HPV16 E6 protein, which maintains the E6 properties in in vitro binding assays. This will be useful for basic studies, and for development of diagnostic kits and immunotherapies in preclinical mouse models of HPV-related tumorigenesis.

Human Papillomaviruses; Epithelial Tropisms, and the Development of Neoplasia.

Papillomaviruses have evolved over many millions of years to propagate themselves at specific epithelial niches in a range of different host species. This has led to the great diversity of papillomaviruses that now exist, and to the appearance of distinct strategies for epithelial persistence. Many papillomaviruses minimise the risk of immune clearance by causing chronic asymptomatic infections, accompanied by long-term virion-production with only limited viral gene expression. Such lesions are typical of those caused by Beta HPV types in the general population, with viral activity being suppressed by host immunity. A second strategy requires the evolution of sophisticated immune evasion mechanisms, and allows some HPV types to cause prominent and persistent papillomas, even in immune competent individuals. Some Alphapapillomavirus types have evolved this strategy, including those that cause genital warts in young adults or common warts in children. These strategies reflect broad differences in virus protein function as well as differences in patterns of viral gene expression, with genotype-specific associations underlying the recent introduction of DNA testing, and also the introduction of vaccines to protect against cervical cancer. Interestingly, it appears that cellular environment and the site of infection affect viral pathogenicity by modulating viral gene expression. With the high-risk HPV gene products, changes in E6 and E7 expression are thought to account for the development of neoplasias at the endocervix, the anal and cervical transformation zones, and the tonsilar crypts and other oropharyngeal sites. A detailed analysis of site-specific patterns of gene expression and gene function is now prompted.

The Nuclear DNA Sensor IFI16 Acts as a Restriction Factor for Human Papillomavirus Replication through Epigenetic Modifications of the Viral Promoters

The human interferon-inducible IFI16 protein, an innate immune sensor of intracellular DNA, was recently demonstrated to act as a restriction factor for human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1) infection by inhibiting both viral-DNA replication and transcription. Through the use of two distinct cellular models, this study provides strong evidence in support of the notion that IFI16 can also restrict human papillomavirus 18 (HPV18) replication. In the first model, an immortalized keratinocyte cell line (NIKS) was used, in which the IFI16 protein was knocked down through the use of small interfering RNA (siRNA) technology and overexpressed following transduction with the adenovirus IFI16 (AdVIFI16) vector. The second model consisted of U2OS cells transfected by electroporation with HPV18 minicircles. In differentiated IFI16-silenced NIKS-HPV18 cells, viral-load values were significantly increased compared with differentiated control cells. Consistent with this, IFI16 overexpression severely impaired HPV18 replication in both NIKS and U2OS cells, thus confirming its antiviral restriction activity. In addition to the inhibition of viral replication, IFI16 was also able to reduce viral transcription, as demonstrated by viral-gene expression analysis in U2OS cells carrying episomal HPV18 minicircles and HeLa cells. We also provide evidence that IFI16 promotes the addition of heterochromatin marks and the reduction of euchromatin marks on viral chromatin at both early and late promoters, thus reducing both viral replication and transcription. Altogether, these results argue that IFI16 restricts chromatinized HPV DNA through epigenetic modifications and plays a broad surveillance role against viral DNA in the nucleus that is not restricted to herpesviruses. Intrinsic immunity is mediated by cellular restriction factors that are constitutively expressed and active even before a pathogen enters the cell. The host nuclear factor IFI16 acts as a sensor of foreign DNA and an antiviral restriction factor, as recently demonstrated by our group for human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1). Here, we provide the first evidence that IFI16 inhibits HPV18 replication by repressing viral-gene expression and replication. This antiviral restriction activity was observed in immortalized keratinocytes transfected with the religated genomes and in U2OS cells transfected with HPV18 minicircles, suggesting that it is not cell type specific. We also show that IFI16 promotes the assembly of heterochromatin on HPV DNA. These changes in viral chromatin structure lead to the generation of a repressive state at both early and late HPV18 promoters, thus implicating the protein in the epigenetic regulation of HPV gene expression and replication.

Acetylation of intragenic histones on HPV16 correlates with enhanced HPV16 gene expression

We report that many histone modifications are unevenly distributed over the HPV16 genome in cervical cancer cells as well as in HPV16-immortalized keratinocytes. For example, H3K36me3 and H3K9Ac that are common in highly expressed cellular genes and over exons, were more common in the early than in the late region of the HPV16 genome. In contrast, H3K9me3, H4K20me3, H2BK5me1 and H4K16Ac were more frequent in the HPV16 late region. Furthermore, a region encompassing the HPV16 early polyadenylation signal pAE displayed high levels of histone H3 acetylation. Histone deacetylase (HDAC) inhibitors caused a 2- to 8-fold induction of HPV16 early and late mRNAs in cervical cancer cells and in immortalized keratinocytes, while at the same time increasing the levels of acetylated histones in the cells and on the HPV16 genome specifically. We concluded that increased histone acetylation on the HPV16 genome correlates with increased HPV16 gene expression.

Identification of HPV integration and gene mutation in HeLa cell line by integrated analysis of RNA-Seq and MS/MS data.

HeLa cell line, which was derived from cervical carcinoma, provides an idea platform to study both the integration of human papillomavirus and the massive mutations occurring on the cancer cell genome. Proteogenomics is a field with the intersection of proteomics and genomics to perform gene annotation and identify gene mutation. In this work, we first identified the SNV/INDEL, structural variation (SV), and virus infection/integration events from RNA-Seq data of HeLa cell line; then, by applying proteogenomics strategy, we were able to detect some of the genomic events with the tandem mass spectrometry (MS/MS) data from the same sample. Furthermore, some of the mutated peptides were experimentally validated using multiple reaction monitoring technology. The integrated analysis of the RNA-Seq and MS/MS data not only renders the discovery of HeLa cell genome variations more credible but also illustrates a practical workflow for protein-coding mutation discovery in cancer-related studies.