HPV16 E7 Genetic Conservation Is Critical to Carcinogenesis.

Although most cervical human papillomavirus type 16 (HPV16) infections become undetectable within 1-2 years, persistent HPV16 causes half of all cervical cancers. We used a novel HPV whole-genome sequencing technique to evaluate an exceptionally large collection of 5,570 HPV16-infected case-control samples to determine whether viral genetic variation influences risk of cervical precancer and cancer. We observed thousands of unique HPV16 genomes; very few women shared the identical HPV16 sequence, which should stimulate a careful re-evaluation of the clinical implications of HPV mutation rates, transmission, clearance, and persistence. In case-control analyses, HPV16 in the controls had significantly more amino acid changing variants throughout the genome. Strikingly, E7 was devoid of variants in precancers/cancers compared to higher levels in the controls; we confirmed this in cancers from around the world. Strict conservation of the 98 amino acids of E7, which disrupts Rb function, is critical for HPV16 carcinogenesis, presenting a highly specific target for etiologic and therapeutic research.

The role of HPV11 E7 in modulating STING-dependent interferon ß response in recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) is a rare benign tumor caused mainly by the infection of the respiratory tract epithelial cells by the human papillomavirus (HPV) type 6/11. However, the specific mechanisms underlying the inhibition of the host's innate immune response by HPV remain unclear. For this purpose, we employed single-cell RNA sequencing to analyze the states of various immune cells in RRP samples post-HPV infection and utilized a cellular model of HPV infection to elucidate the mechanisms by which HPV evades the innate immune system in RRP. The results revealed distinct immune cell heterogeneity in RRP and demonstrated that HPV11 E7 can inhibit the phosphorylation of the stimulator of interferon genes protein, thereby circumventing the body's antiviral response. In vitro co-culture experiments demonstrated that stimulation of macrophages to produce interferon-beta induced the death of HPV-infected epithelial cells, also reducing HPV viral levels. In summary, our study preliminarily identifies the potential mechanisms by which HPV evades the host's antiviral immune response, as well as the latent antiviral functions exhibited by activated macrophages. This research serves as an initial exploration of antiviral immune evasion in RRP, laying a solid foundation for investigating immunotherapeutic approaches for the disease.IMPORTANCESurgical tumor reduction is the most common treatment for recurrent respiratory papillomatosis (RRP). One of the characteristics of RRP is its persistent recurrence, and multiple surgeries are usually required to control the symptoms. Recently, some adjuvant therapies have shown effectiveness, but none of them can completely clear human papillomavirus (HPV) infection, and thus, a localized antiviral immune response is significant for disease control; after all, HPV infection is limited to the epithelium. Inhibition of interferon-beta (IFN-ß) secretion by HPV11 E7 viral proteins in epithelial cells by affecting stimulator of interferon genes phosphorylation may account for the persistence of low-risk HPV replication in the RRP. Moreover, suppression of the IFN-I pathway in RRP cell types might provide clues regarding the hyporeactive function of local immune cells. However, activation of macrophage groups to produce IFN-ß can still destroy HPV-infected cells.

The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer.

The human papillomavirus (HPV) oncoprotein E7 is a relatively short-lived protein required for HPV-driven cancer development and maintenance. E7 is degraded through ubiquitination mediated by cullin 1 (CUL1) and the ubiquitin-conjugating enzyme E2 L3 (UBE2L3). However, E7 proteins are maintained at high levels in most HPV-positive cancer cells. A previous proteomics study has shown that UBE2L3 and CUL1 protein levels are increased by the knockdown of the E3 ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8). We have recently demonstrated that HPV16 upregulates MARCHF8 expression in HPV-positive keratinocytes and head and neck cancer (HPV+ HNC) cells. Here, we report that MARCHF8 stabilizes the HPV16 E7 protein by degrading the components of the S-phase kinase-associated protein 1-CUL1-F-box ubiquitin ligase complex in HPV+ HNC cells. We found that MARCHF8 knockdown in HPV+ HNC cells drastically decreases the HPV16 E7 protein level while increasing the CUL1 and UBE2L3 protein levels. We further revealed that the MARCHF8 protein binds to and ubiquitinates CUL1 and UBE2L3 proteins and that MARCHF8 knockdown enhances the ubiquitination of the HPV16 E7 protein. Conversely, the overexpression of CUL1 and UBE2L3 in HPV+ HNC cells decreases HPV16 E7 protein levels and suppresses tumor growth in vivo. Our findings suggest that HPV-induced MARCHF8 prevents the degradation of the HPV16 E7 protein in HPV+ HNC cells by ubiquitinating and degrading CUL1 and UBE2L3 proteins.IMPORTANCESince human papillomavirus (HPV) oncoprotein E7 is essential for virus replication; HPV has to maintain high levels of E7 expression in HPV-infected cells. However, HPV E7 can be efficiently ubiquitinated by a ubiquitin ligase and degraded by proteasomes in the host cell. Mechanistically, the E3 ubiquitin ligase complex cullin 1 (CUL1) and ubiquitin-conjugating enzyme E2 L3 (UBE2L3) components play an essential role in E7 ubiquitination and degradation. Here, we show that the membrane ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8) induced by HPV16 E6 stabilizes the E7 protein by degrading CUL1 and UBE2L3 and blocking E7 degradation through proteasomes. MARCHF8 knockout restores CUL1 and UBE2L3 expression, decreasing E7 protein levels and inhibiting the proliferation of HPV-positive cancer cells. Additionally, overexpression of CUL1 or UBE2L3 decreases E7 protein levels and suppresses in vivo tumor growth. Our results suggest that HPV16 maintains high E7 protein levels in the host cell by inducing MARCHF8, which may be critical for cell proliferation and tumorigenesis.

BRCA1 deficiency enhances the aggressiveness of breast cancer cells expressing HPV16 oncoproteins.

BACKGROUND INFORMATION: The precise etiology of breast cancer is not completely understood, although women with BRCA1 gene mutations have a significantly increased risk of developing the disease. In addition, sporadic breast cancer is frequently associated with decreased BRCA1 gene expression. Growing evidence of Human papillomaviruses (HPVs) infections in breast tumors has raised the possibility of the involvement of HPVs in the pathogenesis of breast cancer. We investigated whether the effects of HPV oncoproteins E6 and E7 were influenced by the expression levels of BRCA1. HPV16E6E7 (prototype or E6D25E/E7N29S Asian variant type) were stably expressed in MDA-MB231 breast cancer cells, wild type for BRCA1, or with BRCA1 knocked down. RESULTS: Expression of HPV16E6E7 oncogenes did not affect BRCA1 levels and the abundance of HPV16E6E7 was not altered by BRCA1 knockdown. BRCA1 levels did not alter HPV16E6E7-dependent degradation of G1-S cell cycle proteins p53 and pRb. However, we found that the expression of G2-M cell cycle protein cyclin B1 enhanced by HPV16E6E7 was impacted by BRCA1 levels. Especially, we found the correlation between BRCA1 and cyclin B1 expression and this was also confirmed in breast cancer samples from a Thai cohort. We further demonstrated that the combination of HPV oncoproteins and low levels of BRCA1 protein appears to enhance proliferation and invasion. Transactivation activities of HPV16E6E7 on genes regulating cell proliferation and invasion (TGF-ß and vimentin) were significantly increased in BRCA1-deficient cells. CONCLUSIONS: Our results indicate that a deficiency of BRCA1 promotes the transactivation activity of HPV16E6E7 leading to increase of cell proliferation and invasion. SIGNIFICANCE: HPV infection appears to have the potential to enhance the aggressiveness of breast cancers, especially those deficient in BRCA1.

Evidence for direct interaction between the oncogenic proteins E6 and E7 of high-risk human papillomavirus (HPV).

Human papillomaviruses (HPVs) are DNA tumor viruses that infect mucosal and cutaneous epithelial cells of more than 20 vertebrates. High-risk HPV causes about 5% of human cancers worldwide, and the viral proteins E6 and E7 promote carcinogenesis by interacting with tumor suppressors and interfering with many cellular pathways. As a consequence, they immortalize cells more efficiently in concert than individually. So far, the networks of E6 and E7 with their respective cellular targets have been studied extensively but independently. However, we hypothesized that E6 and E7 might also interact directly with each other in a novel interaction affecting HPV-related carcinogenesis. Here, we report a direct interaction between E6 and E7 proteins from carcinogenic HPV types 16 and 31. We demonstrated this interaction via cellular assays using two orthogonal methods: coimmunoprecipitation and flow cytometry-based FRET assays. Analytical ultracentrifugation of the recombinant proteins revealed that the stoichiometry of the E6/E7 complex involves two E7 molecules and two E6 molecules. In addition, fluorescence polarization showed that (I) E6 binds to E7 with a similar affinity for HPV16 and HPV31 (in the same micromolar range) and (II) that the binding interface involves the unstructured N-terminal region of E7. The direct interaction of these highly conserved papillomaviral oncoproteins may provide a new perspective for studying HPV-associated carcinogenesis and the overall viral life cycle.

Field experience with the 8-HPV-type oncoprotein test for cervical cancer screening among HPV-positive women living with and without HIV in LMICs.

Overexpression of HPV-oncoproteins E6 and E7 is necessary for HPV-driven cervical carcinogenesis. Hence, these oncoproteins are promising disease-specific biomarkers. We assessed the technical and operational characteristics of the 8-HPV-type OncoE6/E7 Cervical Test in different laboratories using cervical samples from HPV-positive women living with (WLWH) and without HIV. The 8-HPV-type OncoE6/E7 Test (for short: "OncoE6/E7 test") was performed in 2833 HIV-negative women and 241 WLWH attending multicentric studies in Latin America (ESTAMPA study), and in Africa (CESTA study). Oncoprotein positivity were evaluated at each testing site, according to HIV status as well as type-specific agreement with HPV-DNA results. A feedback questionnaire was given to the operators performing the oncoprotein test to evaluate their impression and acceptability regarding the test. The OncoE6/E7 test revealed a high positivity rate heterogeneity across all testing sites (I2: 95.8%, p < .01) with significant lower positivity in WLWH compared to HIV-negative women (12% vs 25%, p < .01). A similar HPV-type distribution was found between HPV DNA genotyping and oncoprotein testing except for HPV31 and 33 (moderate agreement, k = 0.57). Twenty-one laboratory technicians were trained on oncoprotein testing. Despite operators' concerns about the time-consuming procedure and perceived need for moderate laboratory experience, they reported the OncoE6/E7 test as easy to perform and user-friendly for deployment in resource-limited settings. The high positivity rate variability found across studies and subjectivity in test outcome interpretation could potentially results in oncoprotein false positive/negative, and thus the need for further refinements before implementation of the oncoprotein testing in screen-triage-and-treat approaches is warranted.

Bioinformatics Analysis Reveals E6 and E7 of HPV 16 Regulate Metabolic Reprogramming in Cervical Cancer, Head and Neck Cancer, and Colorectal Cancer through the PHD2-VHL-CUL2-ELOC-HIF-1α Axis.

Human papillomavirus 16 (HPV 16) infection is associated with several types of cancer, such as head and neck, cervical, anal, and penile cancer. Its oncogenic potential is due to the ability of the E6 and E7 oncoproteins to promote alterations associated with cell transformation. HPV 16 E6 and E7 oncoproteins increase metabolic reprogramming, one of the hallmarks of cancer, by increasing the stability of hypoxia-induced factor 1 α (HIF-1α) and consequently increasing the expression levels of their target genes. In this report, by bioinformatic analysis, we show the possible effect of HPV 16 oncoproteins E6 and E7 on metabolic reprogramming in cancer through the E6-E7-PHD2-VHL-CUL2-ELOC-HIF-1α axis. We proposed that E6 and E7 interact with VHL, CUL2, and ELOC in forming the E3 ubiquitin ligase complex that ubiquitinates HIF-1α for degradation via the proteasome. Based on the information found in the databases, it is proposed that E6 interacts with VHL by blocking its interaction with HIF-1α. On the other hand, E7 interacts with CUL2 and ELOC, preventing their binding to VHL and RBX1, respectively. Consequently, HIF-1α is stabilized and binds with HIF-1ß to form the active HIF1 complex that binds to hypoxia response elements (HREs), allowing the expression of genes related to energy metabolism. In addition, we suggest an effect of E6 and E7 at the level of PHD2, VHL, CUL2, and ELOC gene expression. Here, we propose some miRNAs targeting PHD2, VHL, CUL2, and ELOC mRNAs. The effect of E6 and E7 may be the non-hydroxylation and non-ubiquitination of HIF-1α, which may regulate metabolic processes involved in metabolic reprogramming in cancer upon stabilization, non-degradation, and translocation to the nucleus.

An engineered Accum-E7 protein-based vaccine with dual anti-cervical cancer activity.

Worldwide prevalence of cervical cancer decreased significantly with the use of human papilloma virus (HPV)-targeted prophylactic vaccines. However, these multivalent antiviral vaccines are inert against established tumors, which leave patients with surgical ablative options possibly resulting in long-term reproductive complications and morbidity. In an attempt to bypass this unmet medical need, we designed a new E7 protein-based vaccine formulation using Accum™, a technology platform designed to promote endosome-to-cytosol escape as a means to enhance protein accumulation in target cells. Prophylactic vaccination of immunocompetent mice using the Accum-E7 vaccine (aE7) leads to complete protection from cervical cancer despite multiple challenges conducted with ascending C3.43 cellular doses (0.5-, 1.0-, and 2.0 × 106 cells). Moreover, the humoral response induced by aE7 was higher in magnitude compared with naked E7 protein vaccination and displayed potent inhibitory effects on C3.43 proliferation in vitro. When administered therapeutically to animals with pre-established C3.43 or Tal3 tumors, the vaccine-induced response synergized with multiple immune checkpoint blockers (anti-PD-1, anti-CTLA4, and anti-CD47) to effectively control tumor growth. Mechanistically, the observed therapeutic effect requires cross-presenting dendritic cells as well as CD8 T cells predominantly, with a non-negligible role played by both CD4+ and CD19+ lymphocytes. good laboratory practice (GLP) studies revealed that aE7 is immunogenic and well tolerated by immunocompetent mice with no observed adverse effects despite the use of a fourfold exceeding dose. In a nutshell, aE7 represents an ideal vaccine candidate for further clinical development as it uses a single engineered protein capable of exhibiting both prophylactic and therapeutic activity.

Laurencia johnstonii extract reverses early lesions in the K14E7HPV16 murine cervical carcinogenesis model.

Persistent infection with high-risk human papillomavirus (HR-HPV) is a well-established risk factor to the development of cervical intraepithelial neoplasia (CIN), a condition that can progress to cervical cancer (CC) a major health problem worldwide. Recently, there has been growing interest in exploring alternative therapies utilizing natural products, among which is the algae species Laurencia johnstonii Setchell & Gardner, 1924 (L. johnstonii), proposed for the management of precancerous lesions. The aim of this work was to determine the effect of an organic extract from L. johnstonii (ELj) in early cervical lesions (CIN 1). These CIN 1 lesions were generated in a murine model expressing the HR-HPV16 E7 oncoprotein (K14E7HPV transgenic mice) with a single exogenous hormonal stimulus using 17ß-estradiol. The histopathological studies, the determination of cell proliferation and of the apoptotic levels in cervical tissue, showed that, seven doses of ELj (30 mg/kg weight per day diluted in a DMSO-saline solution [1:7]) lead to recovery the architecture of cervical epithelium. Accordingly, in the transgenic mice it was observed a statistically significant decrease of the PCNA expression levels, a marker of cell proliferation, and a statistically significant increase in the apoptosis levels using Caspase 3 as a marker. In addition, we determined the expression levels of the tumor suppressor miR-218 and the oncomiRNA miR-21. Interestingly, our results may suggest that ELj treatment tended to restore the normal expression of both miRNAs as compared with controls being more evident in the non-transgenic induced mice. Differences of p < 0.05 were considered statistically significant through the whole study. Based on these results, we propose that the use of ELj could be an alternative for the treatment of cervical early lesions.

Epitope delimitation: A new method for defining epitopes of human IgG-reactive antigenic peptides based on rabbit-recognized epitope motifs.

The use of precise epitope peptides as antigens is essential for accurate serological diagnosis of viral-infected individuals, but now it remains an unsolvable problem for mapping precise B cell epitopes (BCEs) recognized by human serum. To address this challenge, we propose a novel epitope delimitation (ED) method to uncover BCEs in the delineated human IgG-reactive (HR) antigenic peptides (APs). Specifically, the method based on the rationale of similarities in humoral immune responses between mammalian species consists of a pair of elements: experimentally delineated HR-AP and rabbit-recognized (RR) BCE motif and corresponding pair of sequence alignment analysis. As a result of using the ED approach, after decoding four RR-epitomes of human papillomavirus types 16/18-E6 and E7 proteins utilizing rabbit serum against each recombinant protein and sequence alignment analysis of HR-APs and RR-BCEs, 19 fine BCEs in 17 of 22 known HR-APs were defined based on each corresponding RR-BCE motifs, including the type-specificity of each delimited BCE in homologous proteins. The test with 22 known 16/20mer HR-APs demonstrated that the ED method is effective and efficient, indicating that it can be used as an alternative method to the conventional identification of fine BCEs using overlapping 8mer peptides.

Production of recombinant HPV11/16 E6/E7-MBP-His6 fusion proteins and their potential to induce cytokine secretion by immune cells in peripheral blood.

Human papillomavirus (HPV) infection poses a significant threat to public health worldwide. Targeting the function of HPV E6 and E7 proteins and activating the host immune response against these proteins represent promising therapeutic strategies for combating HPV-related diseases. Consequently, the efficient production of soluble, high-purity E6 and E7 proteins is crucial for function and host immune response studies. In this context, we selected the pMCSG19 protein expression vector for Escherichia coli to produce soluble MBP-His6 tagged HPV11/16 E6/E7 proteins, achieving relatively high purity and yield. Notably, these proteins exhibited low toxicity to peripheral blood mononuclear cells (PBMCs) and did not compromise their viability. Additionally, the recombinant proteins were capable of inducing the secretion of multiple cytokines by immune cells in peripheral blood, indicating their potential to elicit immune responses. In conclusion, our study offers a novel approach for the production of HPV11/16 E6/E7 fusion proteins with relatively high purity and yield. The fusing HPV11/16 E6/E7 proteins to MBP-His6 tag may serve as a valuable method for large-scale protein production in future research endeavors.

Genetic variability of human papillomavirus type 18 based on E6, E7 and L1 genes in central China.

BACKGROUND: High-risk human papillomavirus (HR-HPV) infection is an important factor for the development of cervical cancer. HPV18 is the second most common HR-HPV after HPV16. METHODS: In this study, MEGA11 software was used to analyze the variation and phylogenetic tree of HPV18 E6-E7 and L1 genes. The selective pressure to E6, E7 and L1 genes was estimated using pamlX. In addition, the B cell epitopes of L1 amino acid sequences and T cell epitopes of E6-E7 amino acid sequences in HPV18 were predicted by ABCpred server and IEDB website, respectively. RESULTS: A total of 9 single nucleotide variants were found in E6-E7 sequences, of which 2 were nonsynonymous variants and 7 were synonymous variants. Twenty single nucleotide variants were identified in L1 sequence, including 11 nonsynonymous variants and 9 synonymous variants. Phylogenetic analysis showed that E6-E7 and L1 sequences were all distributed in A lineage. In HPV18 E6, E7 and L1 sequences, no positively selected site was found. The nonconservative substitution R545C in L1 affected hypothetical B cell epitope. Two nonconservative substitutions, S82A in E6, and R53Q in E7, impacted multiple hypothetical T cell epitopes. CONCLUSION: The sequence variation data of HPV18 may lay a foundation for the virus diagnosis, further study of cervical cancer and vaccine design in central China.

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.

The ubiquitin specific protease 7 stabilizes HPV16E7 to promote HPV-mediated carcinogenesis.

Human papillomavirus (HPV) encoded E7 oncoprotein plays an important role in supporting the viral productive cycle and inducing cancer phenotypes. The ability of E7 to exercise these functions, partly, depends upon its steady-state level. HPV manipulates the host de-ubiquitination pathway to maintain the stability of its viral proteins. In this study, we uncovered that HPV interacts with the host ubiquitin specific protease 7 (USP7), a universal de-ubiquitinating enzyme, leading to the stabilization of E7 oncoprotein. We observed that HPV16E7 complexes with USP7 via the E7-CR3 domain, and this E7-USP7 complex exists predominantly in the nucleus. Our results showed that USP7 stabilizes and prolongs the half-life of HPV16E7 by antagonizing ubiquitination and proteasomal degradation. Consistently, when we inhibited USP7 activity using HBX 19818, HPV16E7 protein level was reduced and its turnover was increased. We also provide evidence that HBX 19818-induced USP7 inhibition can halt HPV-mediated carcinogenesis, including cell proliferation, invasion, migration and transformation. These findings indicate that USP7 plays an essential role in stabilizing E7. The specific and potent inhibitory effects of HBX 19818 on HPV-induced carcinogenesis provide a molecular insight, suggesting the potential of targeting USP7 as a new therapeutic approach for the treatment of HPV-associated cancers.

Structural and Dynamic-Based Characterization of the Recognition Patterns of E7 and TRP-2 Epitopes by MHC Class I Receptors through Computational Approaches.

A detailed comprehension of MHC-epitope recognition is essential for the design and development of new antigens that could be effectively used in immunotherapy. Yet, the high variability of the peptide together with the large abundance of MHC variants binding makes the process highly specific and large-scale characterizations extremely challenging by standard experimental techniques. Taking advantage of the striking predictive accuracy of AlphaFold, we report a structural and dynamic-based strategy to gain insights into the molecular basis that drives the recognition and interaction of MHC class I in the immune response triggered by pathogens and/or tumor-derived peptides. Here, we investigated at the atomic level the recognition of E7 and TRP-2 epitopes to their known receptors, thus offering a structural explanation for the different binding preferences of the studied receptors for specific residues in certain positions of the antigen sequences. Moreover, our analysis provides clues on the determinants that dictate the affinity of the same epitope with different receptors. Collectively, the data here presented indicate the reliability of the approach that can be straightforwardly extended to a large number of related systems.

Human papillomavirus type 16 E7 promotes cell viability and migration in cervical cancer by regulating the miR-23a/HOXC8 axis.

BACKGROUND: Human papillomavirus (HPV) is a risk factor for the occurrence of cervical cancer (CC). Here, we aimed to explore the role of HPV16 in CC and identify the underlying mechanism. METHODS: The expression of miR-23a, HPV16 E6/E7 and homeobox C8 (HOXC8) was measured by quantitative real-time PCR or western blot. Cell viability and migration were evaluated using cell counting kit-8, Transwell and wound healing assays. The targeting relationship between miR-23a and HOXC8 was revealed by dual-luciferase reporter assay. RESULTS: miR-23a was downregulated in HPV16-positive (HPV16+) CC tissues and HPV16+ and HPV18+ cells. Additionally, E6/E7 expression was increased in CC cells. Then, we found that E7, rather than E6, positively regulated miR-23a expression. miR-23a suppressed cell viability and migration, whereas E7 overexpression abrogated this suppression. miR-23a targeted HOXC8, which reversed miR-23a-mediated cell viability and migration. CONCLUSIONS: HPV16 E7-mediated miR-23a suppressed CC cell viability and migration by targeting HOXC8, suggesting a novel mechanism of HPV-induced CC.

Cervical cancer (CC) is a common gynaecological malignancy, and persistent human papillomavirus (HPV) infection, especially HPV16, is a main cause of CC. In this study, we explored the role of HPV16 in CC and the molecular mechanism. We used in vitro study to measure CC cell biological behaviours mediated by HPV16 E7, miR-23a and homeobox C8 (HOXC8). We found that HPV16 E7 promotes CC cell viability and migration. miR-23a expression is decreased in CC cells and inhibits cell viability and migration. HOXC8 is a target of miR-23a that reversed the effects on cellular processes caused by miR-23a. These results showed that miR-23a and HOXC8 may be the therapeutic targets of HPV16 E7-infected CC. What is more, our findings provide new insights into the progression of CC.

Mechanical profile of human keratinocytes expressing HPV-18 oncogenes.

During tumorigenesis, the mechanical properties of cancer cells change markedly, with decreased stiffness often accompanying a more invasive phenotype. Less is known about the changes in mechanical parameters at intermediate stages in the process of malignant transformation. We have recently developed a pre-tumoral cell model by stably transducing the immortalized but non-tumorigenic human keratinocyte cell line HaCaT with the E5, E6 and E7 oncogenes from HPV-18, one of the leading causes of cervical cancer and other types of cancer worldwide. We have used atomic force microscopy (AFM) to measure cell stiffness and to obtain mechanical maps of parental HaCaT and HaCaT E5/E6/E7-18 cell lines. We observed a significant decrease in Young's modulus in HaCaT E5/E6/E7-18 cells measured by nanoindentation in the central region, as well as decreased cell rigidity in regions of cell-cell contact measured by Peakforce Quantitative Nanomechanical Mapping (PF-QNM). As a morphological correlate, HaCaT E5/E6/E7-18 cells displayed a significantly rounder cell shape than parental HaCaT cells. Our results therefore show that decreased stiffness with concomitant perturbations in cell shape are early mechanical and morphological changes during the process of malignant transformation.

Repression of Memo1, a Novel Target of Human Papillomavirus Type 16 E7, Increases Cell Proliferation in Cervical Cancer Cells.

Human papillomavirus (HPV)-induced carcinogenesis is associated with unregulated expression of the oncoproteins E6 and E7. HPV E7 is a viral protein that lacks enzymatic activity; however, it can target several cellular proteins to induce cell transformation and promote uncontrolled proliferation. Although several E7 targets have been described, there are still gaps in the understanding of how this oncoprotein drives cells toward malignancy. Here, using a small HPV type 16 (HPV16) E7 peptide in a proteomic approach, we report Memo1 as a new E7 binding partner, interacting through the aspartic and glutamic acid residues (E80 and D81) in the C-terminal region of HPV16 E7. Furthermore, we demonstrate that HPV16 E7 targets Memo1 for proteasomal degradation through a Cullin2-dependent mechanism. In addition, we show that overexpression of Memo1 decreases cell transformation and proliferation and that reduction of Memo1 levels correlate with activation of Akt and an increase in invasion of HPV-positive cervical cancer cell lines. Our results show a novel HPV E7 interacting partner and describe novel functions of Memo1 in the context of HPV-induced malignancy. IMPORTANCE Although numerous targets have been reported to interact with the HPV E7 oncoprotein, the mechanisms involved in HPV-induced carcinogenesis and the maintenance of cell transformation are still lacking. Here, through pulldown assays using a peptide encompassing the C-terminal region of HPV16 E7, we report Memo1 as a novel E7 interactor. High levels of Memo1 correlated with reduced cell proliferation and, concordantly, knockdown of Memo1 resulted in Akt activation in HPV-positive cell lines. These results highlight new mechanisms used by HPV oncoproteins to modulate proliferation pathways in cervical cancer cells and increase our understanding of the link between Memo1 protein and cancer.

Regulation of HPV E7 Stability by E6-Associated Protein (E6AP).

High-risk human papillomaviruses (HPVs) are responsible for most human cervical cancers, and uncontrolled expression of the two key viral oncoproteins, E6 and E7, stimulates the induction of carcinogenesis. Previous studies have shown that both E6 and E7 are closely associated with different components of the ubiquitin proteasome pathway, including several ubiquitin ligases. Most often these are utilized to target cellular substrates for proteasome-mediated degradation, but in the case of E6, the E6AP ubiquitin ligase plays a critical role in controlling E6 stability. We now show that knockdown of E6AP in HPV-positive cervical cancer-derived cells causes a marked decrease in E7 protein levels. This is due to a decrease in the E7 half-life and occurs in a proteasome-dependent manner. In an attempt to define the underlying mechanism, we show that E7 can also associate with E6AP, albeit in a manner different from that of E6. In addition, we show that E6AP-dependent stabilization of E7 also leads to an increase in the degradation of E7's cellular target substrates. Interestingly, ectopic overexpression of E6 oncoprotein results in lower levels of E7 protein through sequestration of E6AP. We also show that increased E7 stability in the presence of E6AP increases the proliferation of the cervical cancer-derived cell lines. These results demonstrate a surprising interplay between E6 and E7, in a manner which is mediated by the E6AP ubiquitin ligase. IMPORTANCE This is the first demonstration that E6AP can directly help stabilize the HPV E7 oncoprotein, in a manner similar to that observed with HPV E6. This redefines how E6 and E7 can cooperate and potentially modulate each other's activity and further highlights the essential role played by E6AP in the viral life cycle and malignancy.

Transcriptomic Landscape of Gene Expression Profiles and Pathways in Juvenile-Onset Recurrent Respiratory Papillomatosis Tumor Tissues and Human Papillomavirus 6 and 11 E6- and E7-Overexpressing Head and Neck Squamous Cell Carcinoma Cell Lines.

Juvenile-onset recurrent respiratory papillomatosis (JORRP) is the most common benign laryngeal neoplasm in children and is considered to be primarily caused by human papillomavirus (HPV) types 6 and 11. In the present study, we performed RNA sequencing (RNA-seq) of 8 tumors and 4 adjacent nontumor tissues to explore the transcriptional profiles of JORRP tumors. A total of 1,151 upregulated genes involved in the interleukin-17 (IL-17) signaling pathway and 1,620 downregulated genes involved in dysregulated inflammatory responses were reported. Immunohistochemistry (IHC) assays confirmed the upregulation of IL-17C in JORRP tumors compared with paired adjacent nontumor tissues. Real-time PCR (RT-PCR) assays showed positive correlations between CXCL1 (CXC chemokine ligands 1) and CXCL8 and the Derkay Clinic Score of JORRP patients. We further overexpressed the HPV6 or HPV11 E6 and E7 oncogenes in SNU-1076 head and neck squamous cell carcinoma (HNSCC) cell lines and carried out RNA-seq. We found that HPV6-E6-E7 gene overexpression resulted in only 16 upregulated genes and 1 downregulated gene; however, HPV11-E6-E7 gene overexpression resulted in 1,776 upregulated genes and 461 downregulated genes compared with the control cell lines. The differentially expressed genes (DEGs) of HPV11-E6-E7 gene overexpression were positively enriched in the DNA replication-related terms by Gene Ontology (GO) analysis and the IL-17 signaling pathway by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Taken together, our present findings revealed IL-17 signaling pathway-related gene profiles that might contribute to disease pathogenesis and that the HPV11 E6 and E7 oncogenes promote disease progression by enhancing tumor growth and activating the IL-17 signaling pathway in JORRP patients. IMPORTANCE Juvenile-onset recurrent respiratory papillomatosis (JORRP) is primarily caused by human papillomavirus 6 (HPV6) and HPV11 infection; however, the gene signatures of tumors are currently less understood. In the present study, we performed RNA sequencing and found upregulated genes associated with the IL-17 signaling pathway and downregulated genes associated with inflammatory-related pathways. Further RNA sequencing was performed in HPV6-E6-E7- or HPV11-E6-E7-overexpressing SNU-1076 HNSCC cells lines to explore the potential pathogenic molecular mechanisms of HPV virus. We found that HPV11-E6-E7 overexpression resulted in gene expression related to DNA replication and the IL-17 signaling pathway. Our results suggested enriched that the IL-17 signaling pathway resulting from HPV11 infection might contribute to JORRP pathogenesis.