HPV-induced Carcinogenesis Research Articles

Genome-Wide Analysis of p53 Targets Reveals SCN2A as a Novel Player in p53-Induced Cell Arrest in HPV-Positive Cells

The host transcription factor p53 is a critical tumor suppressor in HPV-induced carcinogenesis, regulating target genes involved in cell cycle arrest and apoptosis. However, the p53 targets have not been thoroughly analyzed in HPV-infected cells. In this study, p53 signaling in HPV16 and HPV18 cells was activated by depleting the viral oncoprotein E6. Subsequently, p53-regulated genes were identified by comparing them with genes altered in p53-silenced cells. True p53 targets were defined as genes with at least one overlapping p53 binding site and ChIP peak near their locus. Our analysis revealed that while some p53 targets were common to both the HPV16 and HPV18 cells, the majority of the targets differed between these two types, potentially contributing to the varying prevalence of HPV16 and HPV18 in cervical cancer. Additionally, we identified SCN2A as a novel p53 target involved in p53-induced cell cycle arrest in HPV-related carcinogenesis. This study provides new insights into the mechanisms by which p53 inhibits HPV-induced carcinogenesis.

Diagnostic potential of miRNA-135A1 in human papillomavirus associated cervical lesions

Introduction. Human papillomavirus (HPV) infection with high-risk HPVs is an etiological factor in the development of cervical cancer, with HPV type 16 (HPV16) being the most common. The mechanisms leading to disruption of viral oncogene expression and initiation of epithelial cell transformation are poorly understood. Epigenetic regulatory factors, including cellular miRNAs, may play an important role in HPV-induced carcinogenesis, and aberrantly expressed miRNAs may be promising markers for the diagnosis of HPV-associated lesions.Aim. To search for miRNAs involved in the pathogenesis of HPV16-associated cervical cancer and to evaluate their diagnostic potential for the detection of cervical cancer and precancerous lesions.Materials and methods. MiRNA expression in clinical samples was assessed by both next generation sequencing and quantitative stem-loop polymerase chain reaction (sl-qPCR). Plasma miRNAs from patients with precancerous and cancerous lesions and healthy donors were analyzed using sl-qPCR. Loss of heterozygosity in cervical cancer samples was assessed by copy number ratio of MIR135A1 and ACTB genes. A total of 67 patients with cervical cancer, 21 with precancerous cervical lesions and 24 healthy donors were included in the study. The effect of DNA methylation on miRNA-135A1 expression was evaluated after treatment with a demethylating agent of the cervical HPV16-positive SiHa cell line. Changes in the expression of the HPV16 E6 oncogene were analyzed after transfection with synthetic analogues of the mature forms of miRNA-135А1 (miRNA-135a-3p and miRNA-135a-5p).Results. A significant decrease in the expression of miRNA-135A1 and miRNA-135A2 was detected in tumor tissue samples from HPV16-positive cervical cancer, which was confirmed by sl-qPCR in an independent panel of tumor samples. A decrease in miRNA-135A1 expression was shown to result from both loss of heterozygosity of the gene and aberrant DNA methylation. Transfection of mature forms of miRNA-135A1 into SiHa cells resulted in decreased expression of the E6 oncogene of HPV16. Blood plasma samples from patients with cervical cancer and precancerous lesions showed lower levels of miRNA-135a-3p than healthy donors, and ROC analysis indicated its high diagnostic potential.Conclusion. Levels of miRNA-135A1 are significantly reduced in cervical lesions, both in tumor tissue and plasma, and the ability of this miRNA to suppress the expression of the HPV16 E6 oncogene suggests its oncosuppressive properties. Thus, miRNA-135A1 can be used as a promising new marker for the diagnosis of HPV-associated lesions.

Abstract A132: Pathological analysis of a newly established immunocompetent mice with full Hpv16 genome integration

Abstract Human papillomaviruses (HPV) are associated with both benign and malignant epithelial lesions. High-risk HPV subtype HPV 16 is the causative agent of cervical and the majority of oropharyngeal squamous cell carcinomas. Despite the continuous increase in the disease burden, mechanisms and pathways of HPV16-induced cancer growth are not fully understood, thus leading to suboptimal treatment. In last decades, several transgenic mouse models were developed, helped tremendously to study HPV-induced carcinogenesis. However, all existent HPV transgenic mice were targeted early genes expression driven by an artificial promoter and did not encompass the functionality of the whole HPV genome, including the upstream regulatory region (URR) that contains HPV promoters, regulatory elements, and a replication origin, limited our ability to investigate HPV-associated malignant transformation and the role of host factors in this process. Therefore, we generated HPV16 transgenic mouse by using CRISPR/Cas9-mediated genome editing to insert the full-length HPV16 genome into the Rosa26 locus. Two founder mice, with single HPV copy and with tandem HPV integration, were used to establish mouse colonies. Interestingly, regardless of HPV copy number, ~50% of the HPV knock-in mice exhibited early death during the first 2 weeks after birth, while survived pups were significantly smaller than the same age wild-type mice, and had severe hair loss. Intriguingly, the growth retardant and hair loss phenotype in the HPV16 knock-in mice disappeared in ~4 weeks, most likely indicating that viral genes compromise specific stages of mouse development. We evaluated the expression of HPV genes during development, in different mouse organs, and in established fibroblasts and epithelial cell cultures. Our newly established HPV16 knock-in mouse model provides viral gene expression from natural viral promoters offering an essential basis to explore the role of cellular factors in HPV-driven pathologies. Citation Format: Xue Li, Aditi Kothari, Hina Rehmani, Wendell Yarbrough, Natalia Isaeva. Pathological analysis of a newly established immunocompetent mice with full Hpv16 genome integration [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A132.

Inflammation and Stem Cell Stochasticity of HPV-induced Cervical Cancer: Epigenetics Based Biomarkers through Microbiome and Metabolome for Personalized Medicine: A Systematic Review.

Chemoresistance by stemness in HPV-induced cervical carcinogenesis has significant implications for the overall disease-specific survival of the patients. To date, there are no reports related to the implications of significant aspects of inflammation and microbiome-- mediated epigenetics in cervical cancers. The current systematic review delineates the significant aspects of the inflammation-related pathophysiology, cervical cancer diagnosis based on the HPV-indued stemness, and microbiome- mediated epigenetic markers to develop personalized therapies to target the stemness-acquired indefinitely dividing cancer stem cells. We performed a systematic review without a meta- analysis. We searched several public databases, such as Pubmed, ReleMed, National Library of Medicine, and Scopus, related to inflammation, metabolomics, microbiome-mediated epigenetic markers, and HPV-induced stemness. The review significantly described the correlation between microbial inflammation and stem cell stochasticity of HPV-Induced cervical cancer and the expression of epigenetics- based biomarkers through microbiome and metabolome to foster the cervical cancer progression. These are major risk factors that can cause cervical dysplasia with substantial therapy resistance in cervical cancer patients. The qualitative and quantitative examination of the spatial transcriptomic expression of these stemness markers in the dividing cervical cancer stem cells has significant implications in the clinical sector to develop early personalized medicine to prevent cervical precancerous lesions depending on the prognosis of the cervical cancer patients. Mainly, the combinatorial regimen of current therapeutic modalities, along with microbiome-related therapies with future landscape of epigenetics-modulated therapies, may enhance overall disease-specific survival by modulating the stochastic dynamics of basal epithelial cells across the cervical region.

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.

Human Papillomavirus-Associated Tumor Extracellular Vesicles in HPV+ Tumor Microenvironments.

Most infections with human papillomaviruses (HPVs) are self-resolving and asymptomatic. However, some infections can lead to the development of cancer at different mucosal sites, such as the cervix and the head and neck. Head and neck cancers (HNCs) are dichotomized into HPV-positive (HPV+) or HPV-negative (HPV-) based on their respective etiologies. Notably, the tumor microenvironment (TME) of the HPV+ subtype has an immune landscape characterized with increased immune infiltration, higher levels of T cell activation, and higher levels of immunoregulatory stimuli compared to their HPV- counterparts. Both enveloped and nonenveloped viruses hijack the extracellular vesicle (EV) biogenesis pathway to deploy a "trojan horse" strategy with a pseudoviral envelope to enhance infectivity and evade inflammation. EVs derived from HPV-infected tumor cells could allow for the stealth transport of viral cargo to neighboring nonmalignant cellular populations or infiltrating immune cells within the TME. Furthermore, viral cargo or altered cellular cargo from HPV-associated tumor EVs (HPV-TEVs) could alter the functional state or biological responses of the recipient cellular populations, which could shape the distinctive HPV+ TME. This review will cover the impact of EVs released from HPV-infected cells on HPV-induced carcinogenesis, their role in shaping the distinctive HPV+ tumor microenvironment, and current efforts to develop a painless EV-based liquid biopsy for HPV+ cancers.

Abstract LB350: Novel mouse model to study HPV associated pathologies

Abstract Papillomaviruses are species specific, non-enveloped, double-stranded DNA viruses that have an ~8kb circular genome enclosed by a 55 nm icosahedral capsid. Human papillomaviruses (HPV) are associated with both benign and malignant epithelial lesions. High-risk HPV subtypes are the causative agents of cervical and the majority of oropharyngeal squamous cell carcinomas. Persistent HPV infection is required for HPV-associated tumorigenesis; however, cellular factors needed to support the viral persistence and HPV genes expression are not completely understood. Several transgenic mouse models, developed in last decades, helped tremendously to study HPV-induced carcinogenesis. However, the absence of HPV non-coding upstream regulatory region and late coding region, as well as targeted early genes expression driven by an artificial promoter - features that are shared by all existent HPV transgenic mice - limit our ability to investigate HPV-associated malignant transformation and the role of host factors in this process. Therefore, we generated HPV16 transgenic mouse by using CRISPR/Cas9-mediated genome editing to insert the full-length HPV16 genome into the Rosa26 locus. Two founder mice, with single HPV copy and with tandem HPV integration, were used to establish mouse colonies. Interestingly, regardless of HPV copy number, ~30% of the HPV knock-in mice exhibited early death during the first 2 weeks after birth, while survived pups were significantly smaller than the same age wild-type mice, and had severe hair loss. Intriguingly, the growth retardant and hair loss phenotype in the HPV16 knock-in mice disappeared in ~4 weeks, most likely indicating that viral genes compromise specific stages of mouse development. We evaluated the expression of HPV genes during development, in different mouse organs, and in established fibroblasts and epithelial cell cultures. Our newly developed HPV16 knock-in mouse model provides viral gene expression from natural viral promoters offering an essential basis to explore the role of cellular factors in HPV-driven pathologies. Citation Format: Xue Li, Travis Schrank, Aditi Kothari, Hina Rehmani, Jason Tasoulas, Sulgi Kim, Wendell Yarbrough, Natalia Isaeva. Novel mouse model to study HPV associated pathologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB350.

Downregulation of miR-193a/b-3p during HPV-induced cervical carcinogenesis contributes to anchorage-independent growth through PI3K-AKT pathway regulators.

Cervical cancer is caused by a persistent infection with high-risk types of human papillomavirus(HPV) and an accumulation of (epi)genetic alterations in the host cell. Acquisition of anchorage-independent growth represents a critical hallmark during HPV-induced carcinogenesis, thereby yielding the most valuable biomarkers for early diagnosis and therapeutic targets. In a previous study, we found that miR-193a-3p and miR-193b-3p were involved in anchorage-independent growth. This study aimed to delineate the role of miR-193a/b-3p in HPV-induced carcinogenesis and to identify their target genes related to anchorage-independent growth. Cell viability and colony formation were assessed in SiHa cancer cells and HPV-16 and -18 immortalized keratinocytes upon miR-193a/b-3p overexpression. Both microRNAsreduced cell growth of all three cell lines in low-attachment conditions and showed a minor effect in adherent conditions. Online target-predicting programs and publicly available expression data were used to find candidate messenger RNA(mRNA) targets of miR-193a/b-3p. Seven targets showed reduced mRNA expression upon miR-193a/b-3p overexpression. For three targets, Western blot analysis was also performed, all showing a reduced protein expression. A direct interaction was confirmed using luciferase assays for six genes: LAMC1, PTK2, STMN1, KRAS, SOS2, and PPP2R5C, which are phosphatidylinositol 3-kinase/protein kinase B(PI3K-AKT) regulators. All six targets were overexpressed in cervical cancers and/or precursor lesions. Together with an observed downregulation of phosphorylated-AKT upon miR-193a/b-3p overexpression, this underlines the biological relevance of miR-193a/b-3p downregulation during HPV-induced cervical carcinogenesis. In conclusion, the downregulation of miR-193a-3p and miR-193b-3p is functionally involved in the acquisition of HPV-induced anchorage independence by targeting regulators of the PI3K-AKT pathway.

Human papillomavirus 16 E6 promotes angiogenesis of lung cancer via SNHG1.

Human papillomavirus (HPV) is a risk factor for lung cancer. However, the underlying mechanisms are not known. Long noncoding RNAs (lncRNAs) have been found to play an important role in the occurrence and development of lung cancer due to their particular characteristics. HPV-induced lung carcinogenesis is incompletely defined. We aimed to screen and clarify the functions of lncRNAs that are differentially expressed in HPV-related lung cancer. We found that lncRNA SNHG1 is upregulated in lung cancer cells infected with HPV16 E6 by qRT‒PCR. Further results demonstrated that SNHG1 overexpression facilitates the tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Our results also indicated that SNHG1 might function in lung cancer by binding with EGFR. Further studies revealed that SNHG1 overexpression could activate the nuclear factor κb (NF-κB) pathway, which increases the expression of interleukin-6 (IL-6). We also found that IL-6 can activate the STAT3 pathway, which promotes VEGF-D expression. These results expanded our understanding of SNHG1 as a new avenue for therapeutic intervention against lung cancer progression. Upregulation of SNHG1 by HPV infection might be an undefined link between lung cancer and HPV.

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

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

Exosomes Derived from Post-Surgical Drain Fluid of HPV+ Oropharyngeal Cancer Patients Contain HPV DNA and can Induce E6 mRNA Transcription in HPV-Negative Cells

<h3>Purpose/Objective(s)</h3> Human papillomavirus (HPV) infection predisposes to oropharyngeal squamous cell carcinoma (OPC). Exosomes are a component of cell-free DNAs that are actively shed by tumor and immune cells. The role of exosomes in cancer metastasis and in HPV-induced carcinogenesis remains unknown. We hypothesize that exosomes derived from surgical drain fluid (SDF) in HPV+ OPC patients contain HPV viral DNA that can be transmitted directly into HPV-negative cells, resulting in transcription of viral oncogenes. <h3>Materials/Methods</h3> Following surgical neck dissection, SDF samples were collected and processed from 21 HPV+ and 5 HPV- OPC patients (discovery cohort). Exosomes were isolated from SDFs using either the Qiagen exoEasy kit or the SBI SEC EV isolation kit. Exosome structure was characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Immunoblotting was performed to verify exosome-specific biomarkers. Exosomes were pretreated with Dnase (NEB), then exosomal DNA was extracted using a cell-free DNA isolation kit (Qiagen). HPV16 copy number was determined by TaqMan quantitative PCR. Validation was then performed using SDF from a separate cohort of 10 HPV+ OPC patients. To examine transcription of exosome-derived HPV DNA, exosomes were isolated from 3 HPV+ SDF and 1 HPV- SDF. After incubating these exosomes with two HPV- cell lines (SCC22A and SCC25), we isolated total cell line RNA, and performed reverse transcriptase PCR (RT-PCR). <h3>Results</h3> TEM and NTA results revealed rounded structures with a size of 70-150 nm, respectively. Immunoblots detected CD63, CD9, and TSG101 in the protein lysates, indicating the vesicles isolated from SDF were indeed exosomes. In the discovery cohort, HPV16 DNA was detected in SDF exosomes isolated from 81% (n = 17) of patients with HPV+ OPC, while SDF-derived exosomes from all (n=5) HPV- patients had undetectable HPV16 DNA. Importantly, paired comparison of HPV16 levels revealed a significant proportion (46.7±34.4%) of HPV16 cell-free DNA in SDF was contained within exosomes. Results from our validation cohort showed that all the exosome samples (100%) isolated from SDF from 10 HPV+ OPC patients contained HPV DNA, regardless of the exosome isolation method; 24.4±14.4% of HPV16 cell-free DNA was localized in exosomes. Furthermore, an <i>in vitro</i> cell culture assay showed that exosomal HPV16 DNA can undergo uptake by SCC22A and SCC25 cells. RT-PCR of RNA from SCC22A cells incubated with HPV DNA-containing exosomes revealed that exosomal HPV16 DNA can be transcribed into HPV16 E6 mRNA in HPV- recipient cells. <h3>Conclusion</h3> SDF, a novel post-surgical biofluid analyte, is enriched for HPV-containing exosomes from HPV+ OPC patients. Exosomes from these HPV+ OPC patients can transmit viral oncogenes to HPV-negative cells, suggesting a novel mode of virus-mediated spread of disease. Furthermore, exosomal HPV DNA can mediate HPV16 E6 mRNA transcription in HPV- recipient cells, indicating its potential role in OPC tumorigenesis.

Investigation of molecular mechanisms underlying JAK/STAT signaling pathway in HPV-induced cervical carcinogenesis using 'omics' approach.

The precise mechanism of action of Janus Kinases (JAK)/Signal Transducer and activator of Transcription (STAT) signaling in human papillomavirus (HPV)-associated cervical cancer (CaCx) is poorly defined. The present study dissected the underlying components of JAK/STAT signaling in HPV-positive cervical neoplasms. Whole transcriptome profile of CaCx cohort from TCGA database revealed elevated STAT3 and its impact on CaCx patients' survival. Using the RT2 Profiler PCR Array, we analyzed 84 genes of interest associated with JAK/STAT signaling in mRNA derived from HPV-negative and HPV-positive cervical lesions which revealed 21 differentially expressed genes (DEGs). Analyses of DEGs using the Database for Annotation, Visualization and Integrated Discovery tool indicated maximum genes enriched in immune response and negative regulation of apoptotic process. Protein-protein network analysis indicated IL4, STAT5A, STAT4, and JAK3 to be the key genes in the interaction network. Further, 7 key DEGs (IL4R, IRF1, EGFR, OAS1, PIAS1, STAT4, and STAT5A) were validated in TCGA cohort using R2 platform. These genes were differentially expressed among HPV-positive cervical tissues and their correlation with STAT3 was established. EGFR and IL4R showed a comparatively strong correlation with STAT3 that supports their involvement in pathogenesis of CaCx. Finally, the Kaplan-Meier analysis established the prognostic association of the key DEGs, in CaCx cohort. The STAT3 and associated key genes discovered from our study establish a strong pathogenic role of JAK/STAT3 pathway in HPV-mediated cervical carcinogenesis.

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.

Proteases and HPV-Induced Carcinogenesis

Simple SummaryHuman papillomavirus (HPV) infection is a sexually transmitted disease with high prevalence worldwide. Although most HPV infections do not lead to cancer, some HPV types are correlated with the majority of cervical cancers, and with some anogenital and oropharyngeal cancers. Moreover, enzymes known as proteases play an essential role in the pathogenic process in HPV-induced carcinogenesis. This review highlights the role of proteases and recent epidemiological data regarding HPV-dependent carcinogenesis.Persistent infection with Human papillomavirus (HPV) is the main etiologic factor for pre-malignant and malignant cervical lesions. Moreover, HPV is also associated with oropharynx and other anogenital carcinomas. Cancer-causing HPV viruses classified as group 1 carcinogens include 12 HPV types, with HPV 16 and 18 being the most prevalent. High-risk HPVs express two oncoproteins, E6 and E7, the products of which are responsible for the inhibition of p53 and pRB proteins, respectively, in human keratinocytes and cellular immortalization. p53 and pRB are pleiotropic proteins that regulate the activity of several signaling pathways and gene expression. Among the important factors that are augmented in HPV-mediated carcinogenesis, proteases not only control processes involved in cellular carcinogenesis but also control the microenvironment. For instance, genetic polymorphisms of matrix metalloproteinase 1 (MMP-1) are associated with carcinoma invasiveness. Similarly, the serine protease inhibitors hepatocyte growth factor activator inhibitor-1 (HAI-1) and -2 (HAI-2) have been identified as prognostic markers for HPV-dependent cervical carcinomas. This review highlights the most crucial mechanisms involved in HPV-dependent carcinogenesis, and includes a section on the proteolytic cascades that are important for the progression of this disease and their impact on patient health, treatment, and survival.

Expression of LncRNAs in HPV-induced Carcinogenesis and Cancer Cachexia: A Study in K14-HPV16 Mice.

To evaluate the expression of lincRNA-p21, H19, EMX2OS, SNHG12 and MALAT1 in a mouse model of human papillomavirus 16 (HPV16)-induced carcinogenesis and cachexia. Chest skin, ear, tongue, penis and gastrocnemius muscle samples from wild-type mice (HPV-) and K14-HPV16 male mice (HPV+) were collected to evaluate the expression of the selected lncRNAs using real-time PCR (qPCR). In chest skin and ear, H19, SNHG12, EMX2OS and lincRNA-p21 were down-regulated in HPV+ versus HPV- mice. In tongue and penile tissues, there was only down-regulation of lincRNA-p21 in HPV+ mice. Additionally, in penile tissue, lincRNA-p21 expression decreased in HPV-induced lesions comparing with normal tissues. In gastrocnemius muscle, MALAT1 was up-regulated and lincRNA-p21 was down-regulated in HPV+ versus HPV-mice. H19, SNHG12, EMX2OS and lincRNA-p21 may be involved in HPV-induced carcinogenesis. In addition, MALAT1 and lincRNA-p21 may play a role in muscle wasting and contribute to cancer cachexia.

Systemic Delivery of a Dual PI3K/mTOR Inhibitor More Effective than Topical Delivery in Preventing Anal Carcinogenesis in an HPV Transgenic Mouse Model.

Anal dysplasia is a growing health concern that over time can result in squamous cell carcinoma (SqCC) of the anus. In this study, we compare a topical versus systemic (oral) administration of LY3023414, a dual PI3K/mTOR inhibitor, to prevent anal carcinogenesis in a Human Papillomavirus (HPV) mouse model of anal cancer. K14E6/E7 transgenic mice were used to model HPV-induced anal carcinogenesis. Mice with varying starting anal histologies (normal histology, low-grade, and high-grade anal dysplasia) were treated topically at the anus or systemically via oral gavage with LY3023414 with or without topical carcinogen for 20 weeks. Mice were monitored for overt anal tumor development and anal tissue was assessed for histology and markers of PI3K and mTOR activity (pAKT and pS6, respectively). LY3023414 treatment, regardless of the mode of delivery, significantly decreased overt tumor development in mice starting with normal histology and low-grade anal dysplasia. Systemic LY3023414 treatment was more effective in delaying tumor onset than topical treatment. Mice treated with systemic LY3023414 had significantly reduced rates of anal SqCC when starting with normal and low-grade anal dysplasia compared to topical treatment. Topical treatment was only effective in reducing SqCC in the setting of low-grade dysplasia. LY3023414 inhibition of pAKT and pS6 expression varied with starting histology. Neither treatment mode was effective in the setting of high-grade anal dysplasia. Systemic LY3023414 treatment was more effective than topical application in delaying the progression of normal anal histology and low-grade dysplasia to anal cancer in HPV-associated mice.

Polymorphism of TP53 gene and the risk of high human papillomavirus load in cervical epithelial cells

BackgroundCervical cancer is mostly caused by persistent human papillomavirus infection. Host genetic variables, such as polymorphisms, may play a role in the development of cervical cancer and may be a risk factor for HPV-induced carcinogenesis. AimsTo investigate the association between TP53 Pro72Arg polymorphism and the high HPV loads in cervical epithelial cells of women older than 30 years. Subjects and methods154 women infected with HPV and 156 healthy women were recruited into a case–control study. Total DNA was isolated from scrapings of epithelial cells from the cervical canal. Genotyping of the TP53 Pro72Arg polymorphism was carried out using allele-specific PCR. Quantitative analysis of human papillomavirus DNA was performed by the AmpliSens®HPV HCR screen- titre-FRT test system. ResultsNo statistically significant difference was detected between allele and genotype frequencies (p = 0.54 and p = 0.67 respectively) in either women with a medium viral load (3–4 log HPV genomes per 100 thousand human cells) or the control group. The frequencies of the ТР53 Arg72 allele and ТР53 72Arg/Arg genotype were significantly higher in women with a high viral load (above 4 log HPV genomes per 100 thousand human cells) than in the control group (OR = 2.36, p = 0.001 and OR = 2.63, p = 0.002 respectively). The ТР53 Pro72 allele and ТР53 72Pro/Pro genotype are associated with a decreased risk of high HPV load group (OR = 0.42, p = 0.001 and OR = 0.14, p = 0.002 respectively). ConclusionThe risk of developing a high viral load in cervical epithelial cells is associated with the TP53 72Arg allele and the TP53 72Arg/Arg genotype in HPV-positive women.

Genome-Wide Profiling Reveals HPV Integration Pattern and Activated Carcinogenic Pathways in Penile Squamous Cell Carcinoma.

Simple SummaryPenile squamous cell carcinoma (PSCC) has been regarded as an HPV-related cancer for a long time. However, the integration pattern and carcinogenic pathways of HPV in PSCC remain unclear. The results of this study provide insights into the HPV-related carcinogenic mechanism in PSCC, which may be less prone to involvement in the traditional E6/E7 carcinogenic process, and are characterized by effects on the host genome, which result in the inactivation of tumor suppressors (CADM2, etc.) and the activation of oncogenes (KLF5, etc.), thus activating oncogenic signaling pathways (MAPK, JAK/STAT, etc.). This study could enhance our understanding of HPV integration and pave the way for subsequent HPV studies in PSCC.Human papillomavirus (HPV) is a significant etiologic driver of penile squamous cell carcinoma (PSCC). The integration pattern of HPV and its carcinogenic mechanism in PSCC remain largely unclear. We retrospectively reviewed 108 PSCC cases who received surgery between 2008 and 2017. Using high-throughput viral integration detection, we identified 35 HPV-integrated PSCCs. Unlike cervical cancer, the HPV E2 oncogene was not prone to involvement in integration. Eleven of the 35 (31.4%) HPV-integrated PSCCs harbored intact HPV E2; these tumors had lower HPV E6 and E7 expression and higher expression of p53 and pRb proteins than those with disrupted E2 did (p < 0.001 and p = 0.024). Integration breakpoints are preferentially distributed in or near host genes, including previously reported hotspots (KLF5, etc.) and newly identified hotspots (CADM2, etc.), which are mainly involved in oncogenic signaling pathways (MAPK, JAK/STAT, etc.). Regarding the phosphorylation levels of JNK, p38 was higher in HPV-positive tumors with MAPK-associated integration than those in HPV-positive tumors with other integration and those in HPV-negative tumors. In vitro, KLF5 knockdown inhibited proliferation and invasion of PSCC cells, while silencing CADM2 promoted migration and invasion. In conclusion, this study enhances our understanding of HPV-induced carcinogenesis in PSCC, which may not only rely on the E6/E7 oncogenes, but mat also affect the expression of critical genes and thus activate oncogenic pathways.

Lack of Conserved miRNA Deregulation in HPV-Induced Squamous Cell Carcinomas.

Squamous cell carcinomas (SCCs) in the anogenital and head and neck regions are associated with high-risk types of human papillomaviruses (HR-HPV). Deregulation of miRNA expression is an important contributor to carcinogenesis. This study aimed to pinpoint commonly and uniquely deregulated miRNAs in cervical, anal, vulvar, and tonsillar tumors of viral or non-viral etiology, searching for a common set of deregulated miRNAs linked to HPV-induced carcinogenesis. RNA was extracted from tumors and nonmalignant tissues from the same locations. The miRNA expression level was determined by next-generation sequencing. Differential expression of miRNAs was calculated, and the patterns of miRNA deregulation were compared between tumors. The total of deregulated miRNAs varied between tumors of different locations by two orders of magnitude, ranging from 1 to 282. The deregulated miRNA pool was largely tumor-specific. In tumors of the same location, a low proportion of miRNAs were exclusively deregulated and no deregulated miRNA was shared by all four types of HPV-positive tumors. The most significant overlap of deregulated miRNAs was found between tumors which differed in location and HPV status (HPV-positive cervical tumors vs. HPV-negative vulvar tumors). Our results imply that HPV infection does not elicit a conserved miRNA deregulation in SCCs.

The interplay between the vaginal microbiome and innate immunity in the focus of predictive, preventive, and personalized medical approach to combat HPV-induced cervical cancer.

HPVs representing the most common sexually transmitted disease are a group of carcinogenic viruses with different oncogenic potential. The immune system and the vaginal microbiome represent the modifiable and important risk factors in HPV-induced carcinogenesis. HPV infection significantly increases vaginal microbiome diversity, leading to gradual increases in the abundance of anaerobic bacteria and consequently the severity of cervical dysplasia. Delineation of the exact composition of the vaginal microbiome and immune environment before HPV acquisition, during persistent/progressive infections and after clearance, provides insights into the complex mechanisms of cervical carcinogenesis. It gives hints regarding the prediction of malignant potential. Relative high HPV prevalence in the general population is a challenge for modern and personalized diagnostics and therapeutic guidelines. Identifying the dominant microbial biomarkers of high-grade and low-grade dysplasia could help us to triage the patients with marked chances of lesion regression or progression. Any unnecessary surgical treatment of cervical dysplasia could negatively affect obstetrical outcomes and sexual life. Therefore, understanding the effect and role of microbiome-based therapies is a breaking point in the conservative management of HPV-associated precanceroses. The detailed evaluation of HPV capabilities to evade immune mechanisms from various biofluids (vaginal swabs, cervicovaginal lavage/secretions, or blood) could promote the identification of new immunological targets for novel individualized diagnostics and therapy. Qualitative and quantitative assessment of local immune and microbial environment and associated risk factors constitutes the critical background for preventive, predictive, and personalized medicine that is essential for improving state-of-the-art medical care in patients with cervical precanceroses and cervical cancer. The review article focuses on the influence and potential diagnostic and therapeutic applications of the local innate immune system and the microbial markers in HPV-related cancers in the context of 3P medicine.