Human Papillomavirus Gene Expression Research Articles

RNA elements that controlhuman papillomavirus mRNA splicing-targets for therapy?

Human papillomaviruses (HPVs) cause more than 4.5% of all cancer in the world and more than half of these cases are attributed to human papillomavirus type 16 (HPV16). Prophylactic vaccines are available but antiviral drugs are not. Novel targets for therapy are urgently needed. Alternative RNA splicing is extensively used by HPVs to express all their genes and HPV16 is no exception. This process must function to perfection since mis-splicing could perturb the HPV gene expression program by altering mRNA levels or by generating dysfunctional mRNAs. Cis-acting RNA elements on the viral mRNAs and their cognate cellular trans-acting factors control papillomavirus RNA splicing. The precise but delicate nature of the splicing process renders splicing sensitive to interference. As such, papillomavirus RNA splicing is a potential target for therapy. Here we summarize our current understanding of cis-acting HPV16 RNA elements that control HPV16 mRNA splicing via cellular proteins and discuss how they may be exploited as targets for therapy to papillomavirus infections and cancer.

The tumor microenvironment state associates with response to HPV therapeutic vaccination in patients with respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) is a rare, debilitating neoplastic disorder caused by chronic infection with human papillomavirus (HPV) type 6 or 11 and characterized by growth of papillomas in the upper aerodigestive tract. There is no approved medical therapy, and patients require repeated debulking procedures to maintain voice and airway function. PRGN-2012 is a gorilla adenovirus immune-therapeutic capable of enhancing HPV 6/11-specific T cell immunity. This first-in-human, phase 1 study (NCT04724980) of adjuvant PRGN-2012 treatment in adult patients with severe, aggressive RRP demonstrates the overall safety and clinically meaningful benefit observed with PRGN-2012, with a 50% complete response rate in patients treated at the highest dose. Responders demonstrate greater expansion of peripheral HPV-specific T cells compared with nonresponders. Additional correlative studies identify an association between reduced baseline papilloma HPV gene expression, greater interferon responses and expression of CXCL9 and CXCL10, and greater papilloma T cell infiltration in responders. Conversely, nonresponders were characterized by greater HPV and CXCL8 gene expression, increased neutrophilic cell infiltration, and reduced T cell papilloma infiltration. These results suggest that papilloma HPV gene expression may regulate interferon signaling and chemokine expression profiles within the tumor microenvironment that cooperate to govern clinical response to therapeutic HPV vaccination in patients with respiratory papillomatosis.

Introduction of E-Score: A gene expression score to characterize immunogenic versus oncogenic HPV expression in HNSCC.

6057 Background: Human Papillomavirus (HPV) integration in the human genome occurs in a majority of HPV(+) head and neck squamous cell carcinomas (HNSCC), with direct and indirect effects on host gene expression and tumorigenesis. Although previous studies showed that HPV integration in HNSCC is overrepresented in genes often somatically mutated in head and neck, lung, and urogenital cancers, reports are mixed for whether patients with HPV integration have worse clinical outcomes. These disparate results may be due to differences in how integration is defined. Integration can often lead to partial loss of the viral genome (e.g. HPV genes E1, E2, or E5) with retention of the oncogenes E6 and E7, which led to a low E2/E7 expression ratio being used as a biomarker for integration. Conversely, others have used the direct detection of DNA or RNA HPV-human breakpoints to identify tumors harboring integrated HPV. HPV integration and the retention or deletion of HPV genes can dramatically change the expression of HPV genes, and we hypothesize it also affects the host immune response. Hypothesis: Loss of non-oncogenic HPV genes relative to E6 and E7 levels reduces immunogenicity, leading to weakened immune cell infiltration and worse clinical outcomes. Methods: Bulk RNA sequencing data from HPV(+) HNSCC tumors (n = 136) was used to characterize the differences in retention or loss of HPV genes and calculate the E-Score as the sum of the logCPM of HPV genes E1, E2, E4, and E5 relative to E7. Cell type deconvolution was performed using ComBat to correct for batch effects, and CIBERSORTx to quantify cell type proportions within the tumor samples. Kaplan-Meier estimates of recurrence and overall survival (OS) were calculated, and Cox proportional hazards tests with covariates were used to test for association with recurrence and OS. To test the correlation between immune cell type inferred proportions and E-Score, T-Tests were performed between samples with high and low E-scores. Results: Low E-Score was significantly associated with recurrence (p = 0.012) but not with OS (p = 0.39) in the multivariate model accounting for age, tumor stage (AJCC8), smoking status, drinking status, BMI, marital status, and molecular tumor subtype (defined as IMU or KRT). For integration positive samples (n = 66), higher E-Score, suggesting lower rates of HPV gene loss, was correlated with higher percentages of dendritic cells (p = 0.044), macrophages (p = 0.021), and CD8+ T Cells (p = 0.024), but not CD4+ T cells (p = 0.14). This significance did not hold when looking at integration negative samples. Conclusions: These results suggest that HPV+ HNSCC patients with a loss of E1, E2, E4 and E5 tend to have less immune cell infiltration and are more likely to have recurrence. We conclude that E-Score is a potential prognostic biomarker of recurrence in HPV+ HNSCC and potential predictive biomarker for immunotherapy.

Human papilloma virus (HPV) and prostate cancer (PCa): The potential role of HPV gene expression and selected cellular MiRNAs in PCa development

BackgroundProstate cancer (PCa) is one of the most common and health-threatening cancers in men worldwide. The human papillomavirus (HPV) is considered one of the organisms with the potential to be involved in the progression of this cancer. In the present study, we evaluated the association between the expression levels of HPV genes with the expression of selected cellular miRNAs (miR-19a, miR-21, miR-23b, miR-34a, miR-150–5p, and miR-155) and their targets genes (P53, Rb, c-Myc, TIMP-1, MMP-2, MMP-9, PDCD4, Bcl-2, and Survivin) in PCa tissue samples. MethodsHPV detection and genotyping were performed on the tissues of 112 PCa patients and 39 healthy individuals. The expression profile of miRNA was evaluated by SYBR Green-based real-time PCR. As well Human Survivin ELISA Kit was utilized to determine the concentrations of Retinoblastoma, P53, survivin, Bcl-2, c-Myc, TIMP-1, MMP-2, MMP-9, and PDCD4 in the prostate tissues. ResultsAccording to our findings, HPV genome was detected in 28.7% (21/73) of PCa tissue specimens and 17.94% (7/39) control samples. There was no significant association between the presence of HPV infection with PCa (OR = 2.01, 95%CI = 0.8–5.68, P = 0.102). We found that mean expression level of miR-19a (3.7 ± 4.3, p-value: 0.0007), and −21 (2.5 ± 2.8, p-value<0.0001) were significantly higher and miR-23b (−2.14 ± 3.08, p-value: 0.003) and −34a (−3.12 ± 3.28, p-value: 0.0001) levels were significantly lower in PCa tissue samples than in control tissue samples. ConclusionPresent research indicated that HPV positive PCa has a distinct miRNA profile compared with HPV negative PCa.

Lacticaseibacillus casei LH23 Suppressed HPV Gene Expression and Inhibited Cervical Cancer Cells.

Microbiota of lower female reproductive tract is special in its microorganism composition with Lactobacillus as the predominant bacteria. A few of Lactobacillus species have been identified to benefit the inhibition of inflammatory and malignant diseases. Lacticaseibacillus casei LH23 is a strain isolated from traditional fermented food and had been demonstrate to ameliorate DSS-induced colitis in mice. In the present study, effects of Lacticaseibacillus casei LH23 on cervical cancer cells were investigated. Supernatants of lysates and heat-inactivated Lacticaseibacillus casei LH23 were found to inhibit the expression of human papillomavirus genes E6/E7 which is the main causative factor of cervical cancer. With MTT, EdU staining, and TUNEL staining assays, Lacticaseibacillus casei LH23 was shown to suppress the proliferation and induced the apoptosis of cervical cancer cells. Additionally, with wound-healing and Western-blot assays, Lacticaseibacillus casei LH23 was shown to slowdown the migration of cervical cancer cells and altered the expression of metastasis-related genes. These results demonstrated the anti-cervical cancer potential of Lacticaseibacillus casei LH23.

5-aza-2'-deoxycytidine induces apoptosis and inhibits tumour growth in vivo of FaDu cells, a specific HPVnegative HNSCC cell line.

Head and neck cancer squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, resulting in over 600,000 new diagnoses annually. Traditionally, HNCC has been related to tobacco and alcohol exposure; however, over the past decade, a growing number of head and neck cancers are attributed to human papillomavirus (HPV) infection. 5-Aza-2’-deoxycytidine (5-AzaD) was demonstrated as an effective chemotherapeutic agent for acute myelogenous leukaemia. Preclinical data revealed that 5-aza inhibits growth and increases cell death of HPV(+) cancer cells. These effects are associated with reduced expression of HPV genes, stabilization of TP53, and activation of TP53-dependent apoptosis. The aim of the present study is to test the effect of 5-AzaD on growth of human squamous cell carcinoma (FaDu), a HPV(-) and p53 mutated cells, in vitro and in vivo. The effect of 5-AzaD on cell viability, cell cycle progression and induction of apoptosis was tested in vitro. The effect of 5-AzaD on tumour growth in vivo was tested using xenograft mice inoculated with FaDu cells. The results indicated that 5-AzaD reduced cell viability and induced apoptosis in FaDu cells in vitro. In vivo studies revealed that 5-AzaD suppresses the growth of tumours in xenograft mice inoculated with FaDu cells through inhibition of proliferation and induction of apoptosis. These findings may emphasis that 5-AzaD is effective in treatment of HPV(-) HNSCC tumours through TP53 independent pathway. Future studies are needed in order to clarify the molecular mechanism of action of 5-AzaD in HPV(-) cancer cells.

Detection of viral gene expression in risk-stratified biopsies reveals no active HPV in cutaneous squamous cell carcinoma.

Human papillomavirus (HPV) infection is known to promote the development of mucosal squamous cell carcinoma (mSCC), including pathologically high-grade lesions, but its role in cutaneous squamous cell carcinoma (cuSCC) remains unclear, particularly in lesions that are considered high risk. We aimed to determine whether enhanced HPV transcriptional activity can be detected in high-risk cuSCC samples compared with low-grade SCC samples or normal skin. We performed RNA sequencing of cuSCC across 23 risk-stratified skin lesions. A subset of samples was tested for the presence of HPV DNA. High-quality, non-human reads from each sample group were used for viral analysis using Microbiome Coverage Profiler. None of the samples analysed had detectable expression of HPV RNA, while 64% of samples tested positive for HPV DNA. All samples were found to have expression of human endogenous retrovirus, and multiple samples showed expression of other viruses. Viral and prophage gene expression can be monitored in cuSCC or normal skin biopsies, yet no sample in our study showed evidence of active HPV gene expression despite evidence of HPV genome presence. This suggests HPV transcription does not play a role in differentiating high-risk cuSCCs from low-risk cuSCCs or normal skin.

The role of HPV gene expression and selected cellular MiRNAs in lung cancer development

BackgroundThe high mortality rate of lung cancer can be justified that strong need to explore new aspect of tumor biology. Human papillomavirus (HPV) has been detected as risk factor for the development of lung cancer. The aim of this study was to determine the role of HPV and cellular/miRNAs genes expression in the epithelial-mesenchymal transition (EMT) and development of lung cancer. MethodsIn this case-control study, 109 lung cancer tissue and 52 controls were included. We analyzed the presence of HPV infection, its genotypes (in positive samples) and the expression of viral genes (E2, E6 and E7). Also, We examined the expression of celluar factors including (a) p53 and retinoblastoma (Rb) (as anti-carcinogenic genes), (b) EMT related genes, (c) selected miRNAs. ResultsOur results reported 51.4% and 23.1% of HPV genome in tumor tissues and control tissues samples, respectively. There was a significant association between the HPV positive status and lung cancer (OR = 3.26, 95% C.I = 1.47–7.02, P = 0.001). HPV type 16 was the most prevalent genotype in tissues. The expression of p53, RB, TIMP1, CCNG-1, E-cad and PTPN13 were decreased while MMP-2 and N-cad were increased in HPV-positive tumor/control tissues compared to HPV-negative tissues. Also, among miRNAs, let-7, miR-23, miR-34, miR-125, miR-146 were downregulated and miR-20, miR-424 were upregulated in HPV-positve tissues compared to HPV-negative tissues. ConclusionThis study demonstrated that HPV infection and interaction with cellular genes and miRNAs promote EMT which involved in the lung cancer development.

Role of Viral Ribonucleoproteins in Human Papillomavirus Type 16 Gene Expression.

Human papillomaviruses (HPVs) depend on the cellular RNA-processing machineries including alternative RNA splicing and polyadenylation to coordinate HPV gene expression. HPV RNA processing is controlled by cis-regulatory RNA elements and trans-regulatory factors since the HPV splice sites are suboptimal. The definition of HPV exons and introns may differ between individual HPV mRNA species and is complicated by the fact that many HPV protein-coding sequences overlap. The formation of HPV ribonucleoproteins consisting of HPV pre-mRNAs and multiple cellular RNA-binding proteins may result in the different outcomes of HPV gene expression, which contributes to the HPV life cycle progression and HPV-associated cancer development. In this review, we summarize the regulation of HPV16 gene expression at the level of RNA processing with focus on the interactions between HPV16 pre-mRNAs and cellular RNA-binding factors.

The association between HPV gene expression, inflammatory agents and cellular genes involved in EMT in lung cancer tissue

BackgroundLung cancer is a leading cause of cancer morbidity and mortality worldwide. Several studies have suggested that Human papillomavirus (HPV) infection is an important risk factor in the development of lung cancer. In this study, we aim to address the role of HPV in the development of lung cancer mechanistically by examining the induction of inflammation and epithelial-mesenchymal transition (EMT) by this virus.MethodsIn this case-control study, tissue samples were collected from 102 cases with lung cancer and 48 controls. We examined the presence of HPV DNA and also the viral genotype in positive samples. We also examined the expression of viral genes (E2, E6 and E7), anti-carcinogenic genes (p53, retinoblastoma (RB)), and inflammatory cytokines in HPV positive cases.ResultsHPV DNA was detected in 52.9% (54/102) of the case samples and in 25% (12/48) of controls. A significant association was observed between a HPV positive status and lung cancer (OR = 3.37, 95% C.I = 1.58–7.22, P = 0.001). The most prevalent virus genotype in the patients was type 16 (38.8%). The expression of p53 and RB were decreased while and inflammatory cytokines were increased in HPV-positive lung cancer and HPV-positive control tissues compared to HPV-negative lung cancer and HPV-negative control tissues. Also, the expression level of E-cad and PTPN-13 genes were decreased in HPV- positive samples while the expression level of SLUG, TWIST and N-cad was increased in HPV-positive samples compared to negative samples.ConclusionOur study suggests that HPV infection drives the induction of inflammation and EMT which may promote in the development of lung cancer.

Abstract 2130: Human papillomavirus and hepatitis B virus in situ tumor oncoviral genotype and coinfection drive oncogenesis and patient clinical response in cross cancer analysis

Abstract Genotype of hepatitis B virus (HBV) and human papillomavirus (HPV) infection is well known as a significant risk factor in developing liver (HCC), cervical, and head and neck cancers. However, the effect of tumor viral genotype and single-virus coinfection in driving oncogenesis and striating patient survival is generally unclear, as previous studies have not exhaustively genotyped viral-cofactors or profiled viral gene expression in tumors. In this work, we develop and deploy a new scalable, high throughput dynamic sequence alignment tool that facilitates extremely sensitive and precise oncoviral genotype deconvolution using tumor RNA sequencing data from 536 virally infected liver, cervical, and head and neck tumors across several cohorts. Pairing these results with tumor gene expression and survival analyses, we find HBV genotype C (HBV-C) associated HCC patients (n=79) are significantly upregulated in cancer proliferation and decreased cancer survival (FDR&amp;lt;0.01) compared to HBV genotype B associated tumors (n=23). Across 3 liver cancer cohorts, oncogenes TERT, MLL4, and FN1 are found recurrently integrated in HBV-C but not HBV-B. HPV clade a9 infected cervical cancers (n=173) are associated with more aggressive tumor growth and worse survival than HPV clade a7 (n=39) infected tumors (FDR&amp;lt;0.01), and are enriched in COSMIC mutational signatures linked with defective DNA repair and hypermutation. Furthermore, we demonstrate that HPV coinfection may drive patient response as neoantigens from cervical tumors with multiple HPV infections (n=18) are more immunogenic on average than single-infection tumors (n=23), controlling for HPV genotype (p&amp;lt;0.001). Finally, we compare HPV gene expression in cervical and head and neck patients, demonstrating that HPV genotype drives viral gene expression over cancer type (p&amp;lt;0.0001), and indicating viral protein expression may be similar across episomal and integration-based infections. Our results illustrate that HBV and HPV genotype, and coinfection of tumor with multiple genotypes, serve as significant predictors of patient survival and tumor immune responsiveness, and should be considered alongside clinical covariates to improve survival and treatment models. Citation Format: Adrian P. Bubie, Barbara Testoni, Fabien Zoulim, Brett Miles, Marshall Posner, Augusto Villanueva, Bojan Losic. Human papillomavirus and hepatitis B virus in situ tumor oncoviral genotype and coinfection drive oncogenesis and patient clinical response in cross cancer analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2130.

Abstract B14: Variations in the proteasome activity are associated with human papilloma virus (HPV) gene expression and patient survival in HPV+ oropharyngeal tumors

Abstract Variations in the molecular function of human papillomavirus (HPV) have been associated with outcome in HPV-positive oropharyngeal squamous cell carcinomas (HPV+OPSCC). HPV-mediated carcinogenesis is initiated by the E6 and E7 proteins, which promotes the inhibition of the p53 and pRb proteins by targeting them for degradation by the proteasome system. Considering that the proteasome system is an important mediator of HPV molecular activity, we hypothesize that its activity may be an important modifier of HPV+OPSCC phenotypes. To test this hypothesis, we analyzed the in silico expression of proteasome genes among HPV+ oropharyngeal squamous cell carcinoma cases from The Cancer Genome Atlas (TCGA) and explored the proteasome activity of HPV+ squamous cell carcinoma cell lines in vitro. Higher expression of proteasome genes was significantly associated with worse 5-year overall survival (p=0.0003), lower expression levels of E1^E4 gene (p = 0.032), and more HPV integration (p = 0.028). In vitro analysis indicated that proteasome activity was distinct between E1^E4 negative and E1^E4 positive HPV+ cell lines. Interestingly, E1^E4 negative cell lines, which exhibited higher levels of some proteasome genes, were more sensitive to proteasome inhibition than E1^E4 positive cells. These results indicate that proteasome activity may vary significantly among HPV+ OPSCC cases and may represent an important modulator of tumor phenotypes. Expression of proteasome genes and also proteasome activity was highly correlated with expression of HPV genes and may function as a potential predictive biomarker. Finally, proteasome targeting may represent a potential therapeutic alternative for a subgroup of HPV+ OPSCC patients. Citation Format: Frederico O. Gleber-Netto, Meng Gao, Li Shen, Jing Wang, Jeffrey N. Myers, Faye M. Johnson, Curtis R. Pickering. Variations in the proteasome activity are associated with human papilloma virus (HPV) gene expression and patient survival in HPV+ oropharyngeal tumors [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B14.

The Transcriptional Cofactor VGLL1 Drives Transcription of Human Papillomavirus Early Genes via TEAD1.

The TEAD family of transcription factors requires associating cofactors to induce gene expression. TEAD1 is known to activate the early promoter of human papillomavirus (HPV), but the precise mechanisms of TEAD1-mediated transactivation of the HPV promoter, including its relevant cofactors, remain unexplored. Here, we reveal that VGLL1, a TEAD-interacting cofactor, contributes to HPV early gene expression. Knockdown of VGLL1 and/or TEAD1 led to a decrease in viral early gene expression in human cervical keratinocytes and cervical cancer cell lines. We identified 11 TEAD1 target sites in the HPV16 long control region (LCR) by in vitro DNA pulldown assays; 8 of these sites contributed to the transcriptional activation of the early promoter in luciferase reporter assays. VGLL1 bound to the HPV16 LCR via its interaction with TEAD1 both in vitro and in vivo Furthermore, introducing HPV16 and HPV18 whole genomes into primary human keratinocytes led to increased levels of VGLL1, due in part to the upregulation of TEADs. These results suggest that multiple VGLL1/TEAD1 complexes are recruited to the LCR to support the efficient transcription of HPV early genes.IMPORTANCE Although a number of transcription factors have been reported to be involved in HPV gene expression, little is known about the cofactors that support HPV transcription. In this study, we demonstrate that the transcriptional cofactor VGLL1 plays a prominent role in HPV early gene expression, dependent on its association with the transcription factor TEAD1. Whereas TEAD1 is ubiquitously expressed in a variety of tissues, VGLL1 displays tissue-specific expression and is implicated in the development and differentiation of epithelial lineage tissues, where HPV gene expression occurs. Our results suggest that VGLL1 may contribute to the epithelial specificity of HPV gene expression, providing new insights into the mechanisms that regulate HPV infection. Further, VGLL1 is also critical for the growth of cervical cancer cells and may represent a novel therapeutic target for HPV-associated cancers.

Stochastic modeling of human papillomavirusearly promoter gene regulation

High risk forms of human papillomaviruses (HPVs) promote cancerous lesions and are implicated in almost all cervical cancer. Of particular relevance to cancer progression is regulation of the early promoter that controls gene expression in the initial phases of infection and can eventually lead to pre-cancer progression. Our goal was to develop a stochastic model to investigate the control mechanisms that regulate gene expression from the HPV early promoter. Our model integrates modules that account for transcriptional, post-transcriptional, translational and post-translational regulation of E1 and E2 early genes to form a functioning gene regulatory network. Each module consists of a set of biochemical steps whose stochastic evolution is governed by a chemical Master Equation and can be simulated using the Gillespie algorithm. To investigate the role of noise in gene expression, we compared our stochastic simulations with solutions to ordinary differential equations for the mean behavior of the system that are valid under the conditions of large molecular abundances and quasi-equilibrium for fast reactions.The model produced results consistent with known HPV biology. Our simulation results suggest that stochasticity plays a pivotal role in determining the dynamics of HPV gene expression. In particular, the combination of positive and negative feedback regulation generates stochastic bursts of gene expression. Analysis of the model reveals that regulation at the promoter affects burst amplitude and frequency, whereas splicing is more specialized to regulate burst frequency. Our results also suggest that splicing enhancers are a significant source of stochasticity in pre-mRNA abundance and that the number of viruses infecting the host cell represents a third important source of stochasticity in gene expression.

Human Papillomavirus Early Promoter Regulatory Core as a Bistable Switch.

High risk human papillomavirus (HPV) can induce cervical and oropharyngeal cancerous lesions. Different HPV strains, as well as still unknown mechanisms can be associated to a range of biochemical parameters that can importantly affect the HPV gene expression dynamics. For this reason, it is of pivotal importance to investigate how parameters variation can induce interesting behaviors such as viral latency in place of the normal gene replication activity. The aim of this study is to perform bifurcation analysis on a minimal model of the early promoter regulatory core controlled by E2 transcriptional regulation. The bifurcation analysis showed how E2 regulation can induce a bistability on the early promoter gene expression that could explain the interplay between viral latency and gene replication regimen.

CDK9 Inhibitor FIT-039 Suppresses Viral Oncogenes E6 and E7 and Has a Therapeutic Effect on HPV-Induced Neoplasia.

Purpose: Cervical cancer is one of the leading causes of cancer-related deaths among women worldwide. The purpose of this study is to assess the therapeutic effect of the newly developed cyclin-dependent kinase 9 (CDK9) inhibitor FIT-039 on cervical neoplasia induced by human papillomavirus (HPV) infection.Experimental Design: We examined FIT-039 for its effect on HPV gene expression in HPV+ cervical cancer cells. Primary keratinocytes monolayer and organotypic raft culture models were used to evaluate HPV viral replication and cervical intraepithelial neoplasia (CIN) phenotypes. Preclinical pharmacokinetics and toxicity tests for FIT-039 were also conducted. Finally, the anti-HPV effect of FIT-039 was further examined in vivo, using HPV+ cervical cancer xenografts.Results: FIT-039 inhibits HPV replication and expression of E6 and E7 viral oncogenes, restoring tumor suppressors p53 and pRb in HPV+ cervical cancer cells. The therapeutic effect of FIT-039 was demonstrated in CIN model of an organotypic raft culture, where FIT-039 suppressed HPV18-induced dysplasia/hyperproliferation with reduction in viral load. FIT-039 also repressed growth of HPV16+, but not HPV- cervical cancer xenografts without any significant adverse effects. Safety and pharmacokinetics of FIT-039 were confirmed for systemic and topical routes.Conclusions: The CDK9 inhibitor FIT-039 showed potent anti-HPV activity without significant toxicity in preclinical studies. Thus, FIT-039 is expected to be a novel therapeutic for CIN to prevent cervical cancer. Clin Cancer Res; 24(18); 4518-28. ©2018 AACR.

Abstract LB-333: PARP-1 supports HPV infection and HPV-associated head and neck cancer

Abstract Background: As the incidence of human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) continues to rise, mechanistic knowledge of HPV16-driven HNSCC remains incomplete. HPV(+) and HPV(-) HNSCCs are molecularly distinct, with altered signaling pathways that have been characterized by proteomic technologies. We recently reported that poly(ADP-ribose)polymerase-1 (PARP-1) is overexpressed in HPV(+) compared to HPV(-) HNSCC. PARP-1 is a multi-functional protein with the ability to produce polymers of ADP-ribose attached to proteins and with major known roles in maintaining genomic integrity, DNA damage repair, transcription, DNA replication, and cell cycle regulation. However, PARP-1 functions in HPV infection and in head and neck cancer have not been fully uncovered. Here, we examine the role of PARP-1 in HPV infection, the interactions of HPV with cellular proteins, and the therapeutic implications of PARP-1 inhibition in HPV(+) HNSCC. Methods: PARP activity was assessed by immunoblotting for levels of poly(ADP-ribose) and by enzymatic PARP-1 ELISA. Expression of HPV genes was measured by RT-PCR. Murine kidney epithelial cells were established from PARP-1 wild-type and knockout mice, and the absence of PARP-1 expression was confirmed by RT-PCR and immunoblotting. Infection with HPV pseudovirus (PsV) containing a GFP expression plasmid was detected by L1 immunofluorescence staining and GFP detection. Survival studies were done in vitro and in vivo with PARP inhibitors veliparib and olaparib. Results: In addition to being overexpressed, we found that PARP-1 was highly enzymatically active in HPV(+) HNSCC as compared to HPV(-) tumors. HPV16 PsV and major capsid protein L1 activated PARP-1 in vitro and in cells. Depletion of PARP-1 and chemical PARP inhibition repressed HPV16 cellular entry, and infection of cells with HPV PsV increased sensitivity of HPV(-) cells to PARP inhibition. Conclusions: We identified a functional role of PARP-1 in initial HPV infection and in HPV-associated cancer. PARP-1 is activated by HPV major capsid protein L1, and HPV(+) HNSCCs maintain PARP-1 enzymatic activity. On top of its role in initial HPV infection, PARP-1 also appears to be important for survival of HPV-infected cells, as inhibition of PARP-1 sensitized cells to HPV PsV infection. These findings suggest that PARP inhibition may hold significant therapeutic potential in the treatment of HPV-associated HNSCC, with our work resulting in the initiation of a window clinical trial ongoing at the Yale Cancer Center. Citation Format: Cassie Pan, Asel Biktasova, Michael Hajek, Andrew Sewell, Tejas Sathe, Gary Bellinger, Wendell Yarbrough, Natalia Issaeva. PARP-1 supports HPV infection and HPV-associated head and neck cancer [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 LB-333.

STAT3 activation by E6 is essential for the differentiation-dependent HPV18 life cycle.

Human papillomaviruses (HPV) activate a number of host factors to control their differentiation-dependent life cycles. The transcription factor signal transducer and activator of transcription (STAT)-3 is important for cell cycle progression and cell survival in response to cytokines and growth factors. STAT3 requires phosphorylation on Ser727, in addition to phosphorylation on Tyr705 to be transcriptionally active. In this study, we show that STAT3 is essential for the HPV life cycle in undifferentiated and differentiated keratinocytes. Primary human keratinocytes containing high-risk HPV18 genomes display enhanced STAT3 phosphorylation compared to normal keratinocytes. Expression of the E6 oncoprotein is sufficient to induce the dual phosphorylation of STAT3 at Ser727 and Tyr705 by a mechanism requiring Janus kinases and members of the MAPK family. E6-mediated activation of STAT3 induces the transcription of STAT3 responsive genes including cyclin D1 and Bcl-xL. Silencing of STAT3 protein expression by siRNA or inhibition of STAT3 activation by small molecule inhibitors, or by expression of dominant negative STAT3 phosphorylation site mutants, results in blockade of cell cycle progression. Loss of active STAT3 impairs HPV gene expression and prevents episome maintenance in undifferentiated keratinocytes and upon differentiation, lack of active STAT3 abolishes virus genome amplification and late gene expression. Organotypic raft cultures of HPV18 containing keratinocytes expressing a phosphorylation site STAT3 mutant display a profound reduction in suprabasal hyperplasia, which correlates with a loss of cyclin B1 expression and increased differentiation. Finally, increased STAT3 expression and phosphorylation is observed in HPV positive cervical disease biopsies compared to control samples, highlighting a role for STAT3 activation in cervical carcinogenesis. In summary, our data provides evidence of a critical role for STAT3 in the HPV18 life cycle.

The analysis of human papillomavirus DNA in penile cancer tissue by in situ hybridization.

Primary penile cancer is a rare disease. Higher incidence rates occur in underdeveloped countries. Many studies have suggested an association between human papillomavirus (HPV) infection and penile cancer. Although HPV can affect the squamous epithelium of the male genitalia similarly to the female genital tract, the association between penile cancer and HPV remains unclear. In the present study, the HPV gene expression was examined in penile cancer tissue using in situ hybridization (ISH). The present study included 41 cases in which penectomy was performed and 3 cases in which tumor resection was performed to treat pathologically-diagnosed penile cancer at Yokohama City University Medical Center, and its 7 affiliated hospitals between April 1990 and March 2010. The penile cancer tissue was subjected to an ISH analysis, and the clinicopathological features and prognosis were investigated. A total of 5/44 cases (11.4%) showed the expression of high-risk HPV. None of the patients showed the expression of low-risk HPV. The associations between the expression of high-risk HPV, and age, tumor location, tumor size, T stage, pathological differentiation, nuclear grade, Broder's classification, pattern of invasion, Y-K grade, vascular invasion, lymphoid invasion, koilocytosis and lymph-node metastasis were then examined. Patients with a well-differentiated status (P=0.044) and Broder's Grade 1 (P=0.019) showed a significantly lower rate of HPV positivity. The HPV expression was not significantly associated with cancer specific survival (P=0.932). ISH using INFORM HPV III does not detect the HPV genotype, this method is easy to employ and may be useful for the diagnosis of penile cancer tissue, similarly to cervical cancer.

The full transcription map of mouse papillomavirus type 1 (MmuPV1) in mouse wart tissues.

Mouse papillomavirus type 1 (MmuPV1) provides, for the first time, the opportunity to study infection and pathogenesis of papillomaviruses in the context of laboratory mice. In this report, we define the transcriptome of MmuPV1 genome present in papillomas arising in experimentally infected mice using a combination of RNA-seq, PacBio Iso-seq, 5’ RACE, 3’ RACE, primer-walking RT-PCR, RNase protection, Northern blot and in situ hybridization analyses. We demonstrate that the MmuPV1 genome is transcribed unidirectionally from five major promoters (P) or transcription start sites (TSS) and polyadenylates its transcripts at two major polyadenylation (pA) sites. We designate the P7503, P360 and P859 as “early” promoters because they give rise to transcripts mostly utilizing the polyadenylation signal at nt 3844 and therefore can only encode early genes, and P7107 and P533 as “late” promoters because they give rise to transcripts utilizing polyadenylation signals at either nt 3844 or nt 7047, the latter being able to encode late, capsid proteins. MmuPV1 genome contains five splice donor sites and three acceptor sites that produce thirty-six RNA isoforms deduced to express seven predicted early gene products (E6, E7, E1, E1^M1, E1^M2, E2 and E8^E2) and three predicted late gene products (E1^E4, L2 and L1). The majority of the viral early transcripts are spliced once from nt 757 to 3139, while viral late transcripts, which are predicted to encode L1, are spliced twice, first from nt 7243 to either nt 3139 (P7107) or nt 757 to 3139 (P533) and second from nt 3431 to nt 5372. Thirteen of these viral transcripts were detectable by Northern blot analysis, with the P533-derived late E1^E4 transcripts being the most abundant. The late transcripts could be detected in highly differentiated keratinocytes of MmuPV1-infected tissues as early as ten days after MmuPV1 inoculation and correlated with detection of L1 protein and viral DNA amplification. In mature warts, detection of L1 was also found in more poorly differentiated cells, as previously reported. Subclinical infections were also observed. The comprehensive transcription map of MmuPV1 generated in this study provides further evidence that MmuPV1 is similar to high-risk cutaneous beta human papillomaviruses. The knowledge revealed will facilitate the use of MmuPV1 as an animal virus model for understanding of human papillomavirus gene expression, pathogenesis and immunology.