HPV16 E7 modulates the cell surface expression of MET and CD109 via the AP2 complex.

Multiple cellular pathways are affected by HPV E6 and E7 oncoproteins, including endocytic and cellular trafficking. HPV-16 E7 can target the adaptor protein (AP) complex, which contains proteins important during endocytosis transport. To further investigate the role of HPV E7 during this process, we analysed the expression of cell surface proteins in NIKS cells expressing HPV-16 E7. We show that different cell surface proteins are regulated by HPV-16 E7 via interaction with AP2. We observed that the expression of MET and CD109 membrane protein seems to be upregulated in cells expressing E7. Moreover, the interaction of MET and CD109 with AP2 proteins is disrupted by HPV-16 E7. In addition, in the absence of HPV-16 E7, there is a downregulation of the cell membrane expression of MET and CD109 in HPV-positive cell lines. These results expand our knowledge of the functions of E7 and open new potential cellular pathways affected by this oncoprotein.

A paradigm for post-embryonic Oct4 re-expression: E7-induced hydroxymethylation regulates Oct4 expression in cervical cancer.

The Octamer-binding transcription factor-4 (Oct4) is upregulated in different malignancies, yet a paradigm for mechanisms of Oct4 post-embryonic re-expression is inadequately understood. In cervical cancer, Oct4 expression is higher in human papillomavirus (HPV)-related than HPV-unrelated cervical cancers and this upregulation correlates with the expression of the E7 oncogene. We have reported that E7 affects the Oct4-transcriptional output and Oct4-related phenotypes in cervical cancer, however, the underlying mechanism remains elusive. Here, we characterize the Oct4-protein interactions in cervical cancer cells via computational analyses and Mass Spectrometry and reveal that Methyl-binding proteins (MBD2 and MBD3), are determinants of Oct4-driven transcription. E7 triggers MBD2 downregulation and TET1 upregulation, thereby disrupting the methylation status of the Oct4 gene. This coincides with an increase in the total DNA hydroxymethylation leading to the re-expression of Oct4 in cervical cancer and likely affecting broader transcriptional patterns. Our findings reveal a previously unreported mechanism by which the E7 oncogene can regulate Oct4 re-expression and global transcriptional patterns by increasing DNA hydroxymethylation and lowering the barrier to cellular plasticity during carcinogenesis.

Identification and characterisation of novel potential phospho-acceptor sites in HPV-16 E7.

Several studies have described functional regulation of high-risk human papillomaviruses (HPVs), E6 and E7 oncoproteins via posttranslational modifications (PTMs). However, how these PTMs modulate the activity of E6 and E7, particularly in their targeting of cellular proteins, is not completely understood. In this study, we show that HPV16 E7 can be phosphorylated by casein kinase I (CKI) and glycogen synthase kinase 3 (GSK3). This principal phosphorylation occurs at threonine residues 5 and 7 with a more minor role for residues 19-20 in the N-terminal region of 16 E7. Intriguingly, whilst mutational analyses suggest that residues 5 and 7 may be dispensable for the transformation of primary baby rat kidney cells by E7, intact residues 19 and 20 are required. Furthermore, negative charges at these residues (TT19-20DD) enhance the pRb-E7 interaction and cells display increased proliferation and invasion capacities. Using a proteomic approach with a phosphorylated peptide spanning the TT19-20 region of HPV16 E7, we have identified a panel of new, phospho-specific E7 interacting partners. These results shed new light on the complexity of N-terminal phosphorylation of E7 and how this can contribute towards expanding the repertoire of E7 targeted pathways.

KNTC1, regulated by HPV E7, inhibits cervical carcinogenesis partially through Smad2.

Cervical cancer is the second most common malignancy of the female reproductive tract worldwide. Although cervical cancer is caused by human papillomavirus (HPV) infection, its underlying pathogenesis requires further investigation. The present study investigated the role of kinetochore associated protein 1 (KNTC1) in cervical cancer and its association with the key virus oncoprotein, HPV E7. A series of bioinformatic analyses revealed that KNTC1 might be involved in the tumorigenesis of multiple human malignancies, including cervical cancer. Tissue microarray analysis showed that in vivo KNTC1 expression was higher in high-grade squamous intraepithelial lesions (HSILs) than in normal cervix and even higher in cervical cancer. In vitro silencing of KNTC1 increased the proliferation, invasion and migration of cervical cancer cell lines. Although not affecting apoptosis, KNTC1 silencing significantly promoted G1/S phase transition of the cell cycle. High-throughput analysis of mRNA expression showed that KNTC1 could regulate its downstream target protein Smad2 at the transcriptional level. Moreover, as the key oncoprotein of the virus, HPV E7 could inhibit the expression of KNTC1 protein, and decrease Smad2 protein expression with or without the aid of KNTC1. These results indicated that KNTC1 is a novel tumor suppressor that can impede the initiation and progression of cervical carcinoma, providing insight into the molecular mechanism by which HPV induces cervical cancer.

Novel effect of the high risk-HPV E7 CKII phospho-acceptor site on polarity protein expression.

BACKGROUND: Oncogenic Human Papillomaviruses (HPVs) base their transforming potential on the action of both E6 and E7 viral oncoproteins, which perform cooperative or antagonistic actions and thus interfere with a variety of relevant cellular targets. Among them, the expression of some PDZ-containing polarity proteins, as DLG1 and hScrib, is altered during the HPV life cycle and the consequent malignant transformation. Together with the well-established interference of E6 with PDZ proteins, we have recently shown that E7 viral oncoprotein is also responsible for the changes in abundance and localization of DLG1 observed in HPV-associated lesions. Given that the mechanisms involved remained only partially understood, we here thoroughly analyse the contribution of a crucial E7 post-translational modification: its CKII-dependent phosphorylation. Moreover, we extended our studies to hScrib, in order to investigate possible conserved regulatory events among diverse PDZ targets of HPV. METHODS: We have acutely analysed the expression of DLG1 and hScrib in restrictive conditions for E7 phosphorylation by CKII in epithelial culture cells by western blot and confocal fluorescence microscopy. We made use of genome-edited HPV-positive cells, specific inhibitors of CKII activity and transient expression of the viral oncoproteins, including a mutant version of E7. RESULTS: We here demonstrate that the functional phosphorylation of E7 oncoprotein by the CKII cellular kinase, a key regulatory event for its activities, is also crucial to counteract the E6-mediated degradation of the PDZ-polarity protein DLG1 and to promote its subcellular redistribution. Moreover, we show that the CKII-dependent phosphorylation of E7 is able to control the expression of another PDZ target of HPV: hScrib. Remarkably, we found this is a shared feature among different oncogenic HPV types, suggesting a common path towards viral pathogenesis. CONCLUSIONS: The present study sheds light into the mechanisms behind the misexpression of PDZ-polarity proteins during HPV infections. Our findings stress the relevance of the CKII-mediated regulation of E7 activities, providing novel insights into the joint action of HPV oncoproteins and further indicating a conserved and most likely crucial mechanism during the viral life cycle and the associated transformation.

CIGB-300 Peptide Targets the CK2 Phospho-Acceptor Domain on Human Papillomavirus E7 and Disrupts the Retinoblastoma (RB) Complex in Cervical Cancer Cells.

CIGB-300 is a clinical-grade anti-Protein Kinase CK2 peptide, binding both its substrate's phospho-acceptor site and the CK2α catalytic subunit. The cyclic p15 inhibitory domain of CIGB-300 was initially selected in a phage display library screen for its ability to bind the CK2 phospho-acceptor domain ofHPV-16 E7. However, the actual role of this targeting in CIGB-300 antitumoral mechanism remains unexplored. Here, we investigated the physical interaction of CIGB-300 with HPV-E7 and its impact on CK2-mediated phosphorylation. Hence, we studied the relevance of targeting E7 phosphorylation for the cytotoxic effect induced by CIGB-300. Finally, co-immunoprecipitation experiments followed by western blotting were performed to study the impact of the peptide on the E7-pRB interaction. Interestingly, we found a clear binding of CIGB-300 to the N terminal region of E7 proteins of the HPV-16 type. Accordingly, the in vivo physical interaction of the peptide with HPV-16 E7 reduced CK2-mediated phosphorylation of E7, as well as its binding to the tumor suppressor pRB. However, the targeting of E7 phosphorylation by CIGB-300 seemed to be dispensable for the induction of cell death in HPV-18 cervical cancer-derived C4-1 cells. These findings unveil novel molecular clues to the means by which CIGB-300 triggers cell death in cervical cancer cells.

Optimal long peptide for flagellin-adjuvanted HPV E7 cancer vaccine to enhance tumor suppression in combination with anti-PD-1.

Background: Therapeutic cancer vaccines, which induce or amplify tumor-specific T cell responses, are a critical component of multiple combination cancer immunotherapy regimens. Innovative neoantigen identification continually prompts the development of vaccine platforms. However, vaccine monotherapy is not sufficient to eradicate tumors. Thus, therapeutic strategies combining cancer vaccines and treatment with other immune modulators have been expl, ored. Previously, we showed that flagellin has an excellent adjuvant activity to induce effective immune responses to co-administered peptide epitopes through TLR5 stimulation in mouse TC-1 tumor models and flagellin-expressing bacteria modulate the tumor microenvironment (TME) toward enhanced immunogenicity. Methods: Given that short- and long-peptides undergo different fates of internalization, processing, and MHC-restricted presentation by professional antigen-presenting cells (APCs), we compared the antitumor activity of flagellin-adjuvanted peptide vaccines by employing the E7 CD8 epitope short peptide (E7-SP49-57) and E7 long peptides (E7-LP2043-62 and E7-LP3543-77). Because combinations take center stage in immune checkpoint inhibitor (ICI) therapy, we evaluated the best E7 peptide vaccine component for combination with anti-PD-1 in the mouse TC-1 model. Results: Flagellin adjuvanted E7-LP35 vaccine (FlaB-LP35Vax) showed significantly higher antitumor activity than flagellin adjuvanted E7-SP vaccine (FlaB-SPVax) and flagellin adjuvanted E7-LP20 vaccine (FlaB-LP20Vax) in a mouse TC-1 tumor model. Coadministration of flagellin was essential for E7-mediated tumor suppression. PD-1 blockade enhanced the therapeutic efficacy of FlaB-LP35Vax but not FlaB-SPVax. Taken together, E7-LP35 is an optimal tumor antigen for flagellin-adjuvanted E7 cancer vaccines, and the combination of FlaB-LP35Vax with anti-PD-1 antibody treatment induced long-term antitumor immune responses. Conclusions: This result suggests that cooperation between CD4+ and CD8+ cell-mediated immune responses is essential for the success of combination therapy with cancer vaccines and ICIs.

LxCxD motif of the APC/C coactivator subunit FZR1 is critical for interaction with the retinoblastoma protein.

Retinoblastoma protein (pRB) regulates cell cycle by utilizing different regions of its pocket domain for interacting with E2F family of transcription factors and with cellular and viral proteins containing an LxCxE motif. An LxCxE-like motif, LxCxD, is present in FZR1, an adaptor protein of the multi-subunit E3 ligase complex anaphase-promoting complex/cyclosome (APC/C). The APC/CFZR1 complex regulates the timely degradation of multiple cell cycle proteins for mitotic exit and maintains G1 state. We report that FZR1 interacts with pRB via its LxCxD motif. By using point mutations, we found that the cysteine residue in the FZR1 LxCxD motif is critical for direct interaction with pRb. The direct binding of the LxCxD motif of FZR1 to the pRB LxCxE binding pocket is confirmed by using human papillomavirus protein E7 as a competitor, both in vitro and in vivo. While mutation of the cysteine residue significantly disrupts FZR1 interaction with pRB, this motif does not affect FZR1 and core APC/C association. Expression of the FZR1 point mutant results in accumulation of S-phase kinase-associated protein 2 (SKP2) and Polo-like kinase 1 (PLK1), while p27Kip1 and p21Cip1 proteins are downregulated, indicating a G1 cell cycle defect. Consistently, cells containing point mutant FZR1 enter the S phase prematurely. Together our results suggest that the LxCxD motif of FZR1 is a critical determinant for the interaction between FZR1 and pRB and is important for G1 restriction.

Vaccination Strategies for the Control and Treatment of HPV Infection and HPV-Associated Cancer.

Human papillomavirus (HPV) is the most common sexually transmitted infection, currently affecting close to 80 million Americans. Importantly, HPV infection is recognized as the etiologic factor for numerous cancers, including cervical, vulval, vaginal, penile, anal, and a subset of oropharyngeal cancers. The prevalence of HPV infection and its associated diseases are a significant problem, affecting millions of individuals worldwide. Likewise, the incidence of HPV infection poses a significant burden on individuals and the broader healthcare system. Between 2011 and 2015, there were an estimated 42,700 new cases of HPV-associated cancers each year in the United States alone. Similarly, the global burden of HPV is high, with around 630,000 new cases of HPV-associated cancer occurring each year. In the last decade, a total of three preventive major capsid protein (L1) virus-like particle-based HPV vaccines have been licensed and brought to market as a means to prevent the spread of HPV infection. These prophylactic vaccines have been demonstrated to be safe and efficacious in preventing HPV infection. The most recent iteration of the preventive HPV vaccine, a nanovalent, L1-VLP vaccine, protects against a total of nine HPV types (seven high-risk and two low-risk HPV types), including the high-risk types HPV16 and HPV18, which are responsible for causing the majority of HPV-associated cancers. Although current prophylactic HPV vaccines have demonstrated huge success in preventing infection, existing barriers to vaccine acquisition have limited their widespread use, especially in low- and middle-income countries, where the burden of HPV-associated diseases is highest. Prophylactic vaccines are unable to provide protection to individuals with existing HPV infections or HPV-associated diseases. Instead, therapeutic HPV vaccines capable of generating T cell-mediated immunity against HPV infection and associated diseases are needed to ameliorate the burden of disease in individuals with existing HPV infection. To generate a cell-mediated immune response against HPV, most therapeutic vaccines target HPV oncoproteins E6 and E7. Several types of therapeutic HPV vaccine candidates have been developed including live-vector, protein, peptide, dendritic cell, and DNA-based vaccines. This chapter will review the commercially available prophylactic HPV vaccines and discuss the recent progress in the development of therapeutic HPV vaccines.

HPV sensitizes OPSCC cells to cisplatin-induced apoptosis by inhibiting autophagy through E7-mediated degradation of AMBRA1.

Oropharyngeal squamous cell carcinoma (OPSCC) is an increasing world health problem with a more favorable prognosis for patients with human papillomavirus (HPV)-positive tumors compared to those with HPV-negative OPSCC. How HPV confers a less aggressive phenotype, however, remains undefined. We demonstrated that HPV-positive OPSCC cells display reduced macroautophagy/autophagy activity, mediated by the ability of HPV-E7 to interact with AMBRA1, to compete with its binding to BECN1 and to trigger its calpain-dependent degradation. Moreover, we have shown that AMBRA1 downregulation and pharmacological inhibition of autophagy sensitized HPV-negative OPSCC cells to the cytotoxic effects of cisplatin. Importantly, semi-quantitative immunohistochemical analysis in primary OPSCCs confirmed that AMBRA1 expression is reduced in HPV-positive compared to HPV-negative tumors. Collectively, these data identify AMBRA1 as a key target of HPV to impair autophagy and propose the targeting of autophagy as a viable therapeutic strategy to improve treatment response of HPV-negative OPSCC.Abbreviations: AMBRA1: autophagy and beclin 1 regulator 1; CDDP: cisplatin (CDDP); FFPE: formalin-fixed paraffin-embedded (FFPE); HNC: head and neck cancers (HNC); HPV: human papillomavirus (HPV); hrHPV: high risk human papillomavirus (hrHPV); OCSCC: oral cavity squamous carcinomas (OCSSC); OPSCC: oropharyngeal squamous cell carcinoma (OPSCC); OS: overall survival (OS); qPCR: quantitative polymerase chain reaction; RB1: RB transcriptional corepressor 1; ROC: receiver operating characteristic curve (ROC).

Human Papillomavirus E7 Oncoprotein Promotes Proliferation and Migration through the Transcription Factor E2F1 in Cervical Cancer Cells.

BACKGROUND: High-Risk Human Papillomavirus (HR-HPV) persistent infection is the main cause of cervical cancer and its precancerous lesions. A previous study showed that HPV16 and HPV58 infections were the most common infection types in the local region. Some studies also declared that HPV58 E7 variants increased the risk of cervical cancer among Asian populations. OBJECTIVE: This study aimed to determine whether the HPV58 E7 T20I (C632T) variant promotes the malignant behavior of cervical cancer cells and the underlying mechanism of the HR-HPV E7 oncoprotein involved in the development of cervical cancer. METHODS: CCK-8 and clone formation assays were used to detect cell proliferation ability. Transwell assays and cell wound healing assays were used to evaluate cell migration ability. Targeted knockdown of E2F1 expression using specific siRNA, RT-qPCR and Western blot were performed to assess gene expression changes. A chromatin immunoprecipitation assay was used to verify that E2F1 interacted with the TOP2A promoter region. RESULTS: HPV58 E7 and HPV58 E7M oncoproteins increased the proliferation and migration ability of cervical cancer cells. However, the HPV58 E7 T20I variant did not promote malignant behaviors compared with wildtype HPV58 E7. HPV E7 and E7M oncoproteins increased the expression of TOP2A, BIRC5 and E2F1, and knockdown of HPV E7 decreased their expression. Low E2F1 expression reduced the expression of TOP2A and BIRC5 and inhibited the proliferation and migration ability of cervical cancer cells. E2F1 interacted with the TOP2A gene promoter region to promote its transcriptional expression. CONCLUSION: The HPV58 E7 T20I variant did not promote malignant behaviors compared with wild-type HPV58 E7. The HR-HPV E7 oncoprotein enhanced the proliferation and migration of cervical cancer cells, which was considered to be due to the HPV E7 oncoprotein, increasing the expression of BIRC5 and TOP2A by upregulating the transcription factor E2F1.

E7 oncoprotein from human papillomavirus 16 alters claudins expression and the sealing of epithelial tight junctions.

Tight junctions (TJs) are cell­cell adhesion structures frequently altered by oncogenic transformation. In the present study the role of human papillomavirus (HPV) 16 E7 oncoprotein on the sealing of TJs was investigated and also the expression level of claudins in mouse cervix and in epithelial Madin­Darby Canine Kidney (MDCK) cells. It was found that there was reduced expression of claudins ­1 and ­10 in the cervix of 7­month­old transgenic K14E7 mice treated with 17ß­estradiol (E2), with invasive cancer. In addition, there was also a transient increase in claudin­1 expression in the cervix of 2­month­old K14E7 mice, and claudin­10 accumulated at the border of cells in the upper layer of the cervix in FvB mice treated with E2, and in K14E7 mice treated with or without E2. These changes were accompanied by an augmented paracellular permeability of the cervix in 2­ and 7­month­old FvB mice treated with E2, which became more pronounced in K14E7 mice treated with or without E2. In MDCK cells the stable expression of E7 increased the space between adjacent cells and altered the architecture of the monolayers, induced the development of an acute peak of transepithelial electrical resistance accompanied by a reduced expression of claudins ­1, ­2 and ­10, and an increase in claudin­4. Moreover, E7 enhances the ability of MDCK cells to migrate through a 3D matrix and induces cell stiffening and stress fiber formation. These observations revealed that cell transformation induced by HPV16 E7 oncoprotein was accompanied by changes in the pattern of expression of claudins and the degree of sealing of epithelial TJs.

A preliminary study on the immune responses of HPV16-E7 by combined intranasal immunization with lymphotoxin.

OBJECTIVES: Human papillomavirus (HPV) ranks the first cause of cervical cancer. Cervical cancer has high prevalence rates in women around the world. The HPV-E7 oncoprotein is expressed in cervical cancer and is a target of developing immunotherapies against HPV-associated tumors. However, the antigenicity of this protein is low. Due to this reason, potent adjuvants are required to enhance its therapeutic efficacy. This preliminary study aims to evaluate whether lymphotoxin (LT) could act as an effective immune adjuvant for HPV infection in mice models. MATERIAL AND METHODS: Intranasal immunization was used to explore the effect of HPV-E7 and/or LT immune response. After the third intranasal immunization, the titer for the HPV-E7 antibody was detected in serum and vaginal washing fluid. Also, we assessed the expression of chemokine ligand 13 (CXCL13) and Peripheral Node Addressin (PNAd) in the lymph nodes after intranasal immunization with immunohistochemical analysis. RESULTS: compared to HPV-E7 immunization, intranasal immunization with HPV-E7 plus LT significantly increased HPV-E7-specific serum IgG and vaginal IgA titers. Furthermore, the combined use of HPV-E7 and LT strongly induced E7-specific CTL responses. CONCLUSIONS: LT can be effective for intranasal immunized HPV-E7 to improve E7-specific immune responses to HPV infection. It is new approach to eradicate chronic HPV infection capable of inducing an effective anti-infection method.

Inclusion of an E7 DNA Amplification Test Improves the Robustness of Human Papillomavirus-Associated Oropharyngeal Squamous Cell Carcinoma Diagnosis.

BACKGROUND: The rise in human papillomavirus (HPV) infection rates over the last few decades in the USA has contributed to a significant increase in the overall incidence of patients diagnosed with squamous cell carcinoma of the head and neck. These head and neck carcinomas develop in the oropharynx, with more than 90% of them caused by infection with high-risk HPV type 16. Patients diagnosed with HPV-induced oropharyngeal squamous cell carcinomas (OPSCCs) have a better prognosis and treatment response than those diagnosed with head and neck cancers caused by alcohol consumption and tobacco use. To identify patients with HPV-positive OPSCC, new guidelines recommend positive staining of oropharyngeal tissues for p16 INK4a (p16) by immunohistochemistry (IHC). Herein we discuss the testing algorithm that was adopted to address discrepant results between p16 IHC and a DNA in situ hybridization (ISH) test used routinely to diagnose HPV-positive OPSCC patients. METHODS: A DNA polymerase chain reaction (PCR) test that amplifies HPV16 and HPV18 E7 was developed to aid in the diagnosis of HPV-positive OPSCC in a subset of patients. Specimens from these patients stained positive for p16 by an IHC test, but negative for high-risk HPV by a commercial DNA ISH test. Moreover, these results did not match the histopathological characteristics of the specimens, nor the clinical presentations of the patients. RESULTS: Of 21 patients' specimens that were tested for p16 by IHC, 11 specimens showed concordant results with the high-risk HPV 16/18 DNA ISH test. Whereas, in eight p16 IHC positive specimens, HPV viral DNA was not detected by HPV16/18 DNA ISH, and two specimens were not tested by DNA ISH. When these eight p16 IHC positive specimens with discrepant p16 IHC and DNA ISH results were further tested by DNA PCR, six specimens showed concordance with p16 IHC with positive results for HPV16 E7, while two specimens were negative for HPV16 E7 by DNA PCR. All tested specimens were negative for HPV18 E7 by DNA PCR. Thus, the addition of the HPV16 and HPV18 E7 DNA PCR test identified a significant number of false negative test results by the HPV16/18 DNA ISH test and likely several false positive results by p16 IHC. CONCLUSIONS: Inclusion of an HPV16 E7 DNA PCR test improved the robustness of HPV-associated OPSCC diagnosis in patients with discrepant results from p16 IHC staining and a DNA ISH test, and identified patients for proper management with less misclassification.

Vaccination with synthetic long peptide formulated with CpG in an oil-in-water emulsion induces robust E7-specific CD8 T cell responses and TC-1 tumor eradication.

BACKGROUND: Despite considerable efforts at developing therapeutic vaccines for cancer, clinical translation of preclinical successes has been challenging, largely due to the difficulty of inducing strong and sustained cytotoxic T lymphocyte (CTL) responses in patients. Several peptide-based cancer vaccines have failed to show sustainable tumor regression in the clinic, possibly because of a lack of optimization of both the adjuvant and antigen components of the preparations. Here, we aimed to develop and optimize a vaccine format utilizing a synthetic long peptide (SLP) containing the human papilloma virus 16 (HPV16) E7 antigen, with a centrally located defined MHC class I epitope, and evaluate its immunogenicity and efficacy in combination with various adjuvant formulations. METHODS: E731-73 SLP was tested alone or in combination with toll-like receptor (TLR)3, TLR4, TLR7/8 and TLR9 agonists and formulated in oil-in-water (o/w) or water-in-oil (w/o) emulsions to determine a vaccine format inducing a robust CD8 T cell response in murine models. Once a lead vaccine format was determined, we examined its ability to inhibit tumor growth in the murine TC-1 model that expresses HPV16 E7 antigen. RESULTS: We identified the TLR9 agonist CpG formulated in a squalene-based o/w emulsion as the most potent adjuvant, inducing the expansion of multifunctional antigen specific CD8 T cells with cytolytic potential. We also demonstrated that SLP E731-73 + CpG + o/w emulsion vaccine can provide prophylactic and more importantly, therapeutic benefit in the TC-1 murine tumor model. CONCLUSIONS: Our results demonstrate that the novel vaccine format E7 SLP + CpG delivered in an o/w emulsion holds potential for the promotion of strong CTL responses and tumor eradication and encourages further development of peptide/adjuvant vaccines in cancer immunotherapy strategies.

Cervical cancer cell lines are sensitive to sub-erythemal UV exposure.

High risk human papillomavirus (HPV) infections are the causative agent in virtually every cervical cancer as well as a host of other anogenital and oropharyngeal malignancies. These viruses must activate DNA repair pathways to facilitate their replication, while avoiding the cell cycle arrest and apoptosis that can accompany DNA damage. HPV oncoproteins facilitate each of these goals, but also reduce genome stability. Our data dissect the cytotoxic and cytoprotective characteristics of HPV oncogenes in cervical cancer cells. These data show that while the transformation of keratinocytes by HPV oncogene leaves these cells more sensitive to UV, the oncogenes also protect against UV-induced apoptosis. Cisplatin and UV resistant cervical cancer cell lines were generated and probed for their sensitivity to genotoxic agents. Cervical cancer cells can acquire resistance to one DNA crosslinking agent (UV or cisplatin) without gaining broad tolerance of crosslinked DNA. Further, cisplatin resistance may or may not result in sensitivity to PARP1 inhibition.

Aurora kinases are a novel therapeutic target for HPV-positive head and neck cancers.

OBJECTIVES: Human papilloma virus (HPV) is the main culprit in cancers of the cervix, penis, anus, skin, eye and head and neck. Current treatments for HPV cancers have not altered survival outcomes for 30 years and there is a significant lack of targeted therapeutic agents in the management of advanced HPV-related HNSCC. Here we show that survival and maintenance of HPV-positive HNC cells relies on the continuous expression of the major HPV oncogene, E7, and that Aurora kinases are critical for survival of high-risk HPV-positive HNC cells. MATERIALS AND METHODS: To assess the role of HPV E7 on HNC cell survival, RNA interference (RNAi) of the E7 gene was initially performed. Using an Aurora kinase inhibitor, Alisertib, the role of Aurora kinases in the carcinogenesis of HPV E7 positive HNC tumour lines was then investigated. An in vivo HNC xenograft model was also utilised to assess loss of tumour volume in response to RNAi E7 gene silencing and Alisertib treatment. RESULTS: RNAi silencing of the HPV E7 gene inhibited the growth of HPV-positive HNC cells and in vivo tumour load. We show that HPV E7 oncogene expression confers sensitivity to Alisertib on HNC cells where Alisertib-mediated loss in in vitro cell viability and in vivo tumour load is dependent on E7 expression. Moreover, Aurora kinase inhibition induced degradation of MCL-1 in HPV E7-expressing HNC cells. CONCLUSION: Overall, we show that Aurora kinases are a novel therapeutic target for HPV-positive HNCs. It might be feasible to combine Aurora kinase and MCL-1 inhibitors for future HNC therapies.

E7 oncoprotein of human papillomavirus: Structural dynamics and inhibitor screening study.

Human papillomavirus (HPV) has been the primary causative agent of cervical cancer, the most threatening cancer affecting millions of women worldwide. HPV, a small non enveloped DNA virus of high and low risk types contain intrinsically disordered region and it also plays significant role in the development of cervical cancer. HPV E7 contains an ordered Zinc finger motif that binds to pRB and alters its function. It utilizes both disordered N-terminal and structured C-terminal regions for cellular transformation. In this study, we have focused extensively on the evolutionary relationships of E7 among various HPV types and generated a 3D homology model of full length HPV 16 E7, since the structure have not been solved till date. We also analysed the stable conformation and atomic flexibility of modelled E7 through molecular dynamics simulation at 100 ns. To understand the disordered based binding sites of E7 oncoprotein, Molecular recognition features (MoRFs) analysis was carried out on the E7 oncoprotein. The validated model was taken forward for the identification of potential lead compounds and the most prominent compounds were selected for the molecular dynamics simulation of the 100 ns for the stability analysis. Overall, this study highlights the holistic E7 regions including important disordered based binding sites analysed through the MoRFs. The potential inhibitor compound that targets the structured C-terminal region of E7 oncoprotein were subjected for the pharmacological properties analysis and further validated for the binding modes of the compounds with the target structure. This study helps in providing a better intuition to develop a potent anti-HPV agent.

Therapeutic DNA Vaccines for Human Papillomavirus and Associated Diseases.

Human papillomavirus (HPV) has long been recognized as the causative agent of cervical cancer. High-risk HPV types 16 and 18 alone are responsible for over 70% of all cases of cervical cancers. More recently, HPV has been identified as an etiological factor for several other forms of cancers, including oropharyngeal, anogenital, and skin. Thus, the association of HPV with these malignancies creates an opportunity to control these HPV lesions and HPV-associated malignancies through immunization. Strategies to prevent or to therapeutically treat HPV infections have been developed and are still pushing innovative boundaries. Currently, commercial prophylactic HPV vaccines are widely available, but they are not able to control established infections or lesions. As a result, there is an urgent need for the development of therapeutic HPV vaccines, to treat existing infections, and to prevent the development of HPV-associated cancers. In particular, DNA vaccination has emerged as a promising form of therapeutic HPV vaccine. DNA vaccines have great potential for the treatment of HPV infections and HPV-associated cancers due to their safety, stability, simplicity of manufacturability, and ability to induce antigen-specific immunity. This review focuses on the current state of therapeutic HPV DNA vaccines, including results from recent and ongoing clinical trials, and outlines different strategies that have been employed to improve their potencies. The continued progress and improvements made in therapeutic HPV DNA vaccine development holds great potential for innovative ways to effectively treat HPV infections and HPV-associated diseases.

High risk human papilloma viruses (HPVs) are present in benign prostate tissues before development of HPV associated prostate cancer.

BACKGROUND: Although high risk HPVs are associated with an increased risk of prostate cancer it is not known if they have a causal role. The purpose of this study is to investigate the potential role of human papilloma viruses (HPVs) in prostate cancer. The aims are (i) to investigate the presence and confirm the identity of high risk HPVs in benign prostate tissues prior to the development of HPV positive prostate cancer in the same patients, and (ii) to determine if HPVs are biologically active. METHODS: We used polymerase chain reaction (PCR) to identify HPVs in specimens from 52 Australian men with benign prostate biopsies who 1 to 10 years later developed prostate cancer. Immunohistochemistry (IHC) was used to assess the expression of HPV E7 oncoproteins, cytokeratin and prostate specific antigen (PSA). We used RNASeq data from The Cancer Genome Atlas (TCGA) to identify possible HPV RNA sequences in prostate cancer. RESULTS: HPV screening using standard PCR was conducted on 28 of the 52 sets of benign and later prostate cancers. HPV L1 genes were identified in 13 (46%) benign and 8 (29%) of 28 later prostate cancers in the same patients. HPV E7 genes were identified in 23 (82%) benign and 19 (68%) of 28 subsequent prostate cancers in the same patients. The same HPV types were present in both the benign and subsequent prostate cancers in 9 sets of specimens. HPV type 16 was identified in 15% of benign and 3% of prostate cancers. HPV type 18 was identified in 26% of benign and 16% of prostate cancers. Small numbers of HPV types 45, 47, 76 and 115 were also identified. High confidence RNA-Seq evidence for high risk HPV types 16 and 18 was identified in 12 (2%) of the 502 TCGA prostate cancer transcriptomes. High risk HPV E7 oncoprotein was positively expressed in 23 (82%) of 28 benign prostate specimens but only in 8 (29%) of 28 of the later prostate cancer specimens. This difference is statistically significant (p = 0.001). Prostate specific antigen (PSA) was more highly expressed in 26 (50%) of 52 prostate cancer specimens as compared to prior benign prostate specimens in the same patients. CONCLUSIONS: High risk HPVs are present in benign prostate tissues prior to the development of HPV positive prostate cancer. There is a significantly higher expression of HPV E7 oncoproteins in benign prostate tissues as compared to late prostate cancer that subsequently developed in the same patients. This observation suggests that HPV oncogenic activity is an early phenomenon in a majority of prostate oncogenesis. TCGA RNA-Seq data suggests that HPV is biologically active in some prostate tumour samples.