E6 Oncogenes Research Articles

HPV16 E7 Nucleotide Variants Found in Cancer-Free Subjects Affect E7 Protein Expression and Transformation.

Simple SummaryHuman papillomaviruses (HPV) are one of the deadliest viruses causing cancer resulting in over 300,000 deaths annually in the world. HPV16 is the most oncogenic form of HPV and is responsible for over half of all HPV-driven tumors. The viral E7 gene encodes a powerful oncoprotein that can cause infected cells to have uncontrolled growth. A previous study showed that HPV16 isolates with amino acid alterations in the E7 gene were nearly always found in women with HPV infection but no cancer. To study the role of these variants, we expressed them in cultured cells. Most variants produced lower levels of protein, and selected variants had reduced activity in allowing cells to grow in assays that measure tumor cell invasiveness. The data indicate that inhibition of E7 could be a practical approach to treating HPV-driven cancers.The human papillomavirus (HPV) type 16 E7 oncogene is critical to carcinogenesis and highly conserved. Previous studies identified a preponderance of non-synonymous E7 variants amongst HPV16-positive cancer-free controls compared to those with cervical cancer. To investigate the function of E7 variants, we constructed full-length HPV16 E7 genes and tested variants at positions H9R, D21N, N29S, E33K, T56I, D62N, S63F, S63P, T64M, E80K, D81N, P92L, and P92S (found only in controls); D14E, N29H cervical intraepithelial neoplasia (CIN2), and P6L, H51N, R77S (CIN3). We determined the steady-state level of cytoplasmic and nuclear HPV16 E7 protein. All variants from controls showed a reduced level of E7 protein, with 7/13 variants having lower protein levels. In contrast, 2/3 variants from the CIN3 precancer group had near-wild type E7 levels. We assayed the activity of representative variants in stably transfected NIH3T3 cells. The H9R, E33K, P92L, and P92S variants found in control subjects had lower transforming activity than D14E and N29H variants (CIN2), and the R77S (CIN3) had activity only slightly reduced from wild-type E7. In addition, R77S and WT E7 caused increased migration of NIH3T3 cells in a wound-healing assay compared with H9R, E33K, P92L, and P92S (controls) and D14E (CIN2). These data provide evidence that the E7 variants found in HPV16-positive cancer-free women are partially defective for transformation and cell migration, further demonstrating the importance of fully active E7 in cancer development.

Human papillomavirus-related neoplasia of the ocular adnexa.

Human papillomaviruses (HPV) are involved in approximately 5% of solid cancers worldwide. The mucosotropic genotypes infect the stratified epithelium of various locations, where persistent infection may lead to invasive carcinomas. While the causative role of HPV in certain anogenital and head and neck carcinomas is well established, the role of HPV in carcinomas arising in the mucosal membranes of the ocular adnexal tissue (the lacrimal drainage system and the conjunctiva) has been a topic of great uncertainty. Therefore, we conducted a series of studies to assess the correlation between HPV and carcinomas arising in the mucosa of the ocular adnexal tissue and characterize the clinical, histopathological, and genomic features of the tumors in the context of HPV status in a Danish nationwide cohort. We collected clinical and histopathological data and tumor specimens from patients with carcinomas of the conjunctiva and the lacrimal drainage system, and their potential precursors, identified in Danish nationwide registries. The HPV status of the tumors was determined by the combined use of HPV DNA polymerase chain reaction (PCR), HPV E6/E7 mRNA in-situ hybridization, and p16 immunohistochemistry. The genomic profile was investigated by high-throughput DNA sequencing targeting 523 cancer-relevant genes. The literature to date on carcinomas of the lacrimal drainage system and the conjunctiva was summarized. In the Danish cohort, 67% of all carcinomas of the lacrimal drainage system and 21% of all conjunctival carcinomas were HPV-positive. HPV16 was the most frequently implicated genotype. A full-thickness expression of the viral oncogenes E6 and E7 was evident in almost all HPV DNA-positive cases. The HPV-positive carcinomas of the conjunctiva and the lacrimal drainage system shared histopathological and genomic features distinct from their HPV-negative counterparts. The HPV-positive carcinomas were characterized by a non-keratinizing morphology, p16 overexpression, high transcriptional activity of HPV E6/E7, and frequent pathogenic variants in the PI3K-AKT signaling cascade. In contrast, the HPV-negative carcinomas were characterized by a keratinizing morphology, lack of p16 and E6/E7 expression, and frequent somatic pathogenic variants in TP53, CDKN2A, and RB1. Among the patients with conjunctival tumors, HPV positivity was associated with a younger age at diagnosis and a higher risk of recurrence. In conclusion, the results support an etiological role of HPV in a subset of conjunctival and LDS carcinomas and their precursor lesions. Our investigations have shown that the HPV-positive carcinomas of the ocular adnexa share genomic and phenotypic characteristics with HPV-positive carcinomas of other anatomical locations. Therefore, these patients may be eligible for inclusion in future basket trials and future treatment regimens tailored to the more frequently occurring HPV-positive carcinomas of other locations. Future research will further elucidate the diagnostic, prognostic, and predictive role of HPV in these carcinomas.

Genetic deletion of HPV E7 oncogene effectively regresses HPV driven oral squamous carcinoma tumour growth

The major HPV oncogenes, E6 and E7, are known for its notoriety in driving the carcinogenic process in human papilloma virus (HPV) driven cancers. It is well-established that the removal of E7 dampens HPV cancer cell growth and proliferation. This has made E7 an attractive target for HPV cancers. Seminal work from our laboratory employed a CRISPR editing approach to delete E7 which resulted in the effective elimination of HPV+ cervical cancer tumours in vivo. We have also successfully delayed HPV+ tumour growth in vivo with aurora kinase (AURK) inhibitors, an effect which is strongly sensitized by the presence of E7. Unlike our previous observations in cervical cancer cells, in vitro targeting of E6/E7 have only resulted in partial killing of HPV+ oral squamous carcinoma (OSC) cells. However, the effect of sustained removal of E7 on HPV+ OSC tumour growth have not been explored. In this study, we investigated a staggered combination of aurora kinase inhibition, using alisertib, followed by CRISPR editing of E7, to determine if this would lead to better HPV+ OSC killing. Remarkably, genetic deletion of E7 alone was sufficient to effectively regress established HPV+ OSC tumours in vivo suggesting that E7 is essential in the maintenance of HPV+ OSC cancers.

Correlation between HPV PCNA, p16, and p21 Expression in Lung Cancer Patients

Purpose. Evaluate if human papillomavirus (HPV) infection in lung cancer patients might be helping cancer development by altering p16, p21, and PCNA, key human genes involved in cell proliferation and tumor development. Methods. 63 fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) samples from lung tumor patients were used to detect HPV by PCR, followed by genotype through sequencing. The host gene expressions of p21, p16, and PCNA were quantified by qPCR in both FF and FFPE samples, and the expression of viral oncogenes E5, E6, and E7 was also measured by qPCR in 19 FF samples. Results. 74.6% of samples were positive for HPV, 33/44 FFPE samples and 14/19 FF samples. HPV-16 and HPV-18 were detected in 31/33 and 7/33 FFPE, respectively, and HPV-16 was the only type in FF samples. E5, E6, and E7 were expressed in 10/19, 2/19, and 4/19 FF samples, respectively. The p16 RNAm expression was higher in FF HPV+ samples and FFPE+FF HPV+ samples, while p21 showed higher expression in all HPV- samples. In turn, the PCNA expression was higher in HPV+ FF samples; however, in FFPE and FFPE+FF samples, PCNA was higher in HPV- samples. In FF samples, PCNA, p16, and p21 showed a significant positive correlation as well as E5 and E7, and E5 was inversely correlated to p21. In FFPE, also, a positive correlation was observed between PCNA HPV+ and p21 HPV+ and PCNA HPV+ and p16 HPV. In FF+FFPE analysis, a direct correlation was found between PCNA HPV+ and p21 HPV+, p21 HPV+ and p16 HPV+, and PCNA HPV- and p16 HPV-, and an inverse correlation between PCNA HPV+ and p16 HPV+. Also, the p16 protein was positive in 10 HPV+ samples and 1 HPV-. Conclusions. Our data show that lung cancer patients from Northeast Brazil have a high prevalence of HPV, and the virus also expresses its oncogenes and correlates with key human genes involved in tumor development. This data could instigate the development of studies focused on preventive strategies, such as vaccination, used as a prognostic indicator and/or individualized therapy.

Expression profiling of mRNA and functional network analyses of genes regulated by human papilloma virus E6 and E7 proteins in HaCaT cells.

Human papillomavirus (HPV) oncogenes E6 and E7 are essential for HPV-related cancer development. Here, we developed a cell line model using lentiviruses for transfection of the HPV16 oncogenes E6 and E7 and investigated the differences in mRNA expression during cell adhesion and chemokine secretion. Subsequently, RNA sequencing (RNA-seq) analysis was performed to explore the differences in mRNA expression. Compared to levels in the control group, 2,905 differentially expressed mRNAs (1,261 downregulated and 1,644 upregulated) were identified in the HaCaT-HPV16E6E7 cell line. To predict the functions of these differentially expressed genes (DEGs) the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases were used. Protein–protein interactions were established, and the hub gene was identified based on this network. Real-time quantitative-PCR (RT-qPCR) was conducted to confirm the levels of 14 hub genes, which were consistent with the RNA-seq data. According to this, we found that these DEGs participate in the extracellular matrix (ECM), cell adhesion, immune control, and cancer-related signaling pathways. Currently, an increasing number of clinicians depend on E6/E7mRNA results to make a comprehensive judgment of cervical precancerous lesions. In this study, 14 hub genes closely related to the expression of cell adhesion ability and chemokines were analyzed in HPV16E6E7-stably expressing cell lines, which will open up new research ideas for targeting E6E7 in the treatment of HPV-related cancers.

Identification regulatory noncoding RNAs of human papilloma virus type 16 (Papillomaviridae: Alphapapillomavirus: Human papillomavirus) in cervical tumors

High carcinogenic-risk human papillomaviruses (hrHPVs) are recognized as etiological agents of cervical cancer. Constant expression of the viral oncoproteins, E6 and E7, is required for maintenance of the malignant phenotype of tumor cells. The exact mechanism of regulation of viral oncogenes expression in tumor cells is not fully elucidated. identification of viral noncoding RNAs (ncRNAs) in HPV16-positve cervical cancer. The reverse transcription polymerase chain reactions were used to detect viral ncRNAs in HPV16-positve primary cervical squamous cell carcinomas and SiHa and CasKi cell lines. The knockdown technique with oligonucleotides complementary to ncRNAs was used to elucidate their functions. We have identified ncRNAs transcribed in the upstream regulatory region of HPV16 in the cervical carcinoma cell lines and in 32 out 32 cervical squamous cell carcinomas with episomal or integrated forms of HPV16 DNA. Knockdown of sense or antisense strains of ncRNAs by oligonucleotides results in a decrease or increase of the E6 and E7 oncogenes mRNA levels in cells, respectively. These changes of oncogenes mRNA levels are accompanied by the modulation of the levels of the p53 protein, the main target of the E6 oncoprotein. The presence of regulatory ncRNAs in all examined tumors and cell lines revealed for the first time indicates their necessity for maintenance of constant expression of E6 and E7 oncogenes in them. The findings can be useful for understanding of the fundamental aspects of the viral expression regulation in HPV16-positive tumors.

The Interaction of Human Papillomavirus Infection and Prostaglandin E2 Signaling in Carcinogenesis: A Focus on Cervical Cancer Therapeutics

Chronic infection by high-risk human papillomaviruses (HPV) and chronic inflammation are factors associated with the onset and progression of several neoplasias, including cervical cancer. Oncogenic proteins E5, E6, and E7 from HPV are the main drivers of cervical carcinogenesis. In the present article, we review the general mechanisms of HPV-driven cervical carcinogenesis, as well as the involvement of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) and downstream effectors in this pathology. We also review the evidence on the crosstalk between chronic HPV infection and PGE2 signaling, leading to immune response weakening and cervical cancer development. Finally, the last section updates the current therapeutic and preventive options targeting PGE2-derived inflammation and HPV infection in cervical cancer. These treatments include nonsteroidal anti-inflammatory drugs, prophylactic and therapeutical vaccines, immunomodulators, antivirals, and nanotechnology. Inflammatory signaling pathways are closely related to the carcinogenic nature of the virus, highlighting inflammation as a co-factor for HPV-dependent carcinogenesis. Therefore, blocking inflammatory signaling pathways, modulating immune response against HPV, and targeting the virus represent excellent options for anti-tumoral therapies in cervical cancer.

Human Papillomavirus 16 E6 and E7 Oncoproteins Alter the Abundance of Proteins Associated with DNA Damage Response, Immune Signaling and Epidermal Differentiation.

The high-risk human papillomaviruses are oncogenic viruses associated with almost all cases of cervical carcinomas, and increasing numbers of anal, and oral cancers. Two oncogenic HPV proteins, E6 and E7, are capable of immortalizing keratinocytes and are required for HPV associated cell transformation. Currently, the influence of these oncoproteins on the global regulation of the host proteome is not well defined. Liquid chromatography coupled with quantitative tandem mass spectrometry using isobaric-tagged peptides was used to investigate the effects of the HPV16 oncoproteins E6 and E7 on protein levels in human neonatal keratinocytes (HEKn). Pathway and gene ontology enrichment analyses revealed that the cells expressing the HPV oncoproteins have elevated levels of proteins related to interferon response, inflammation and DNA damage response, while the proteins related to cell organization and epithelial development are downregulated. This study identifies dysregulated pathways and potential biomarkers associated with HPV oncoproteins in primary keratinocytes which may have therapeutic implications. Most notably, DNA damage response pathways, DNA replication, and interferon signaling pathways were affected in cells transduced with HPV16 E6 and E7 lentiviruses. Moreover, proteins associated with cell organization and differentiation were significantly downregulated in keratinocytes expressing HPV16 E6 + E7. High-risk HPV E6 and E7 oncoproteins are necessary for the HPV-associated transformation of keratinocytes. However their influence on the global dysregulation of keratinocyte proteome is not well documented. Here shotgun proteomics using TMT-labeling detected over 2500 significantly dysregulated proteins associated with E6 and E7 expression. Networks of proteins related to interferon response, inflammation and DNA damage repair pathways were altered.

O-GlcNAcylation stabilizes the autophagy-initiating kinase ULK1 by inhibiting chaperone-mediated autophagy upon HPV infection

Human papillomaviruses (HPVs) cause a subset of head and neck squamous cell carcinomas (HNSCCs). Previously, we demonstrated that HPV16 oncogene E6 or E6/E7 transduction increases the abundance of O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT), but OGT substrates affected by this increase are unclear. Here, we focus on the effects of O-GlcNAcylation on HPV-positive HNSCCs. We found that upon HPV infection, Unc-51-like kinase 1 (ULK1), an autophagy-initiating kinase, is hyper-O-GlcNAcylated, stabilized, and linked with autophagy elevation. Through mass spectrometry, we identified that ULK1 is O-GlcNAcylated at Ser409, which is distinct from the previously reported Thr635/Thr754 sites. It has been demonstrated that PKCα mediates phosphorylation of ULK1 at Ser423, which attenuates its stability by shunting ULK1 to the chaperone-mediated autophagy (CMA) pathway. Using biochemical assays, we demonstrate that ULK1 Ser409Ser410 O-GlcNAcylation antagonizes its phosphorylation at Ser423. Moreover, mutations of Ser409A and its neighboring site Ser410A (2A) render ULK1 less stable by promoting interaction with the CMA chaperone HSC70 (heat shock cognate 70 kDa protein). Furthermore, ULK1-2A mutants attenuate the association of ULK1 with STX17, which is vital for the fusion between autophagosomes and lysosomes. Analysis of The Cancer Genome Atlas (TCGA) database reveals that ULK1 is upregulated in HPV-positive HNSCCs, and its level positively correlates with HNSCC patient survival. Overall, our work demonstrates that O-GlcNAcylation of ULK1 is altered in response to environmental changes. O-GlcNAcylation of ULK1 at Ser409 and perhaps Ser410 stabilizes ULK1, which might underlie the molecular mechanism of HPV-positive HNSCC patient survival.

HPV16-E6/E7 Oncogene Mutation and p53 Expression among Indonesian Women with Cervical Cancer.

To characterize HPV16 E6/E7 mutation and its association with p53 expression among Indonesian women with cervical cancer. This is a cross-sectional study involving 31 Indonesian women with pathologically proven cervical cancer and HPV16 infection. Data about the clinical characteristics of the study population were obtained from the medical records. Biopsy specimen of the cervical cancer mass from each study participant was obtained for DNA isolation. The ORFs of E6 and E7 genes were amplified using specific primer designed according to K02718/HPV16R gene sequence obtained from GenBank. Sequencing was performed using software program MEGA10. HPV16 E6 and E7 prototype sequences for nucleotide alignment (HPv16. P, GenBank Access code: NC_001526) was selected from European variant. The sequence of nucleotide and amino acid was aligned using software program BioEdit. p53 expression was evaluated through immunohistochemistry and quantified using immunoreactivity score (IRS). Twelve subjects (38.7%) present with E6 and E7 mutation. Median age, parity, stage and histologic type of the tumour did not associate with E6/E7 mutation. E6 and E7 mutation rate was 25.8% (8/31) and 12.9% (4/31), respectively. Seven single nucleotide changes were identified within the E6 and E7 oncogenes, including four non-synonymous and three synonymous mutations. E6 T27C was the most prevalent mutation (16.1%). Nonsynonymous mutations were more prevalent within E7 gene (9.6%) (N29T, N29S, and R77C). Median IRS did not differ between HPV16-E6/E7 variants and wildtype (p value = 0.990). There was no association between E6/E7 mutations and p53 expression in Indonesian women with cervical cancer (PR 1.4, 95% CI: 0.29-6.77, p value = 0.704). HPV16 E6 mutation was more prevalent than E7 mutation among Indonesian women. There was no association between E6/E7 mutation and p53 expression level.

Longitudinal follow-up of HPV16 sequence after cervical infection: Low intrahost variation and no correlation with clinical evolution.

Human papillomavirus (HPV) 16 exhibits different variants that may differ in their carcinogenic risk. To identify some high-risk variants, we sequenced and compared HPV16 whole genomes obtained from a longitudinal cohort of 34 HPV16-infected women who had either spontaneously cleared their infection (clearance group or "C"), or developed cervical high-grade lesions following a viral persistence (group persistence or "P"). Phylogenetic analysis of paired samples obtained at the beginning (C0 or P0) and at the end (C2 or P2) of the follow-up (median intervals between C0-C2 and between P0-P2 were 16 and 36.5 months, respectively) revealed a low genetic variability within the host compared to the genetic interhost diversity. By comparing our HPV16 sequences to a reference sequence, we observed 301 different substitutions, more often transitions (60.9%) than transversions (39.1%), that occurred throughout the viral genome, but with a low frequency in E6 and E7 oncogenes (10 and 9 substitutions), suggesting a high conservation of these genes. Deletions and insertions were mostly observed in intergenic regions of the virus. The only significant substitution found between the subgroups C2 and P2 was observed in the L2 gene (L330F), with an unclear biological relevance. Our results suggest a low longitudinal intrahost evolution of HPV16 sequences and no correlation between genetic variations and clinical evolution.

An overview of the general characteristics, pathogenicity, transmission, and diagnosis of human papilloma virus (HPV)

Mucous membrane infections are brought on by the double-standard DNA virus known as human papillomavirus (HPV). Cervix cell alterations are caused by a sexually transmitted illness. E6 and E7 oncogenes play a critical role in HPV infection. Finding these genes to identify HPV strains, particularly the HPV16 strain, will have a significant impact due to its exceptional sensitivity, the dielectric electrochemical biosensor stands out among other pathogen detection methods. Recent evidence suggests that E6 and E7 are also important in inhibiting the innate immune response of the host cell to HPV. Viral replication is mediated by the E1 and E2 proteins in conjunction with other biological stimuli. E2 has also been linked to viral and cellular transcriptional regulation. Despite decades of research, the function of other viral proteins still remains unclear. Therefore, analysis of Human Papilloma Virus (HPV) characteristics, pathogenicity, transmission and diagnosis were reviewed. We concluded that we now have the tools and methodologies necessary to answer this critical question about viral tropism. The discovery of efficient treatments to cure or prevent HPV-induced illness is just as vital as studying the underlying mechanisms of HPV pathogenesis. The identification of drugs specifically to treat HPV infection has not been highly successful due to the complexities of the HPV life cycle and the limited number of enzymatic activities identified for HPV proteins. The development of drug treatments for existing HPV disease is an important undertaking that deserves further attention. In this regard, the development of therapeutic vaccines and self-protection are promising area of investigation and needs to be further supported.

HnRNP E1 Regulates HPV16 Oncogene Expression and Inhibits Cervical Cancerization.

hnRNP E1 (heterogeneous nuclear ribonucleoprotein E1) is an important RNA-binding protein (RBPs) that plays a vital role in tumor development. Human papillomavirus 16 (HPV16) contains numerous sites that can bind to RNA/DNA and may be modified by multiple RBPs, which contribute to HPV gene expression and HPV-associated cancer development. However, the effects of hnRNP E1 on HPV16 oncogenes in the development of cervical lesions remain unclear. A total of 816 participants with different grades of cervical lesions were enrolled in a community-based cohort established in Shanxi Province, China. The Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases were used to analyze the association between hnRNP E1 mRNA expression and cervical lesions. Cells with up_ and down_regulated hnRNP E1 were established. hnRNP E1 functions were evaluated using cell counting kit-8, flow cytometry analyses, and chromatin immunoprecipitation sequencing. Our results showed that hnRNP E1 expression was linearly dependent on the severity of the cervical lesions. Low expression of HPV16 E2, high expression of E6, and a low ratio of E2 to E6 could increase the risk of cervical lesions. hnRNP E1 expression was correlated with HPV16 oncogene expression. hnRNP E1-relevant genes were involved in the dopaminergic synapses, Wnt signaling pathway, gnRH secretion, and mTOR signaling pathway. hnRNP E1 significantly inhibited cell proliferation, induced apoptosis, arrested the cell cycle at the G0/G1 stage, and decreased HPV16 E6 expression. Our results indicate that hnRNP E1 could downregulate HPV16 E6 oncogene expression and inhibit cervical cancerization, which sheds new light on preventing the carcinogenicity of HPV across a range of diseases by regulating RNA-binding proteins.

Abstract 3284: HB-201 and HB-202, an arenavirus-based immunotherapy, induces tumor T cell infiltration in patients with HNSCC and other HPV16+ tumors

Abstract Background: Human Papillomavirus 16 (HPV16) is the most common etiologic agent of HPV-associated cancers. Treatment options are limited for patients with HPV16+ recurrent or metastatic cancers. HPV16 E7 and E6 oncogenes constitute attractive immunotherapeutic targets. HB-201 and HB-202 are live-attenuated replicating vectors based on LCMV and PICV arenaviruses, respectively, that express the same non-oncogenic HPV16 E7E6 fusion protein for induction of tumor specific T cell responses. Methods: A Phase I/II open labelled clinical trial of HB-201 single vector therapy and HB-202/HB-201 alternating two-vector therapy in patients with treatment-refractory HPV16+ cancers is currently ongoing (NCT04180215). To assess circulating E6/E7 specific T cell responses, IFN-γ enzyme-linked immunospot (ELISpot) and intracellular cytokine staining were performed on pre- and post-treatment peripheral blood mononuclear cells (PBMCs). Evaluation of T cell infiltration and PD-L1 status in tumor bed was performed on paired tissue biopsies from a subset of patients by multiplex immunofluorescence. Results: Single vector therapy (HB-201) and alternating two-vector therapy (HB-202/HB-201) rapidly induce high E6/E7 specific T cell levels, reaching up to ~40% of the circulating CD8+ T cell pool. Alternating two-vector therapy seems to maintain E6/E7 specific T cell responses better in continuous dosing compared to single vector therapy. Furthermore, we demonstrate tumor tissue T cell infiltration in more than 50% of patient samples analyzed. Currently, in depth sequencing of paired biopsies is underway to characterize the tumor microenvironment in response to HB-201 and HB-202/HB-201 treatment. Conclusion: In this updated dataset, we show that HB-201 and HB-202/HB-201, rapidly induce unprecedented E6/E7 specific T cell levels in circulation following a single dose. Furthermore, these data are seen in conjunction with a pronounced increase of post-treatment CD8+ T cells in tumor, suggesting E6/E7 specific T cell infiltration. Our arenavirus vectors expressing the E7E6 fusion antigen demonstrate an attractive and safe therapy for patients with treatment refractory HPV16+ cancers. The ability of replicating arenavirus vectors to incorporate a broad range of antigens and the potent T cell inducing capacity provide a strong rationale to apply this novel therapy to other cancers. Citation Format: Donna Edwards, Michael Schwendinger, Kia Katchar, Katia Schlienger, Klaus Orlinger, Igor Matushansky, Henning Lauterbach. HB-201 and HB-202, an arenavirus-based immunotherapy, induces tumor T cell infiltration in patients with HNSCC and other HPV16+ tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3284.

Abstract 5919: Regulation of SLPI secretion by NFX1-123 and HPV 16 E6 in cervical cancer

Abstract Background: Secretory Leukocyte Peptidase Inhibitor (SLPI) functions in cancer progression, cellular invasion, metastasis, immune evasion, and chemoresistance. Our previous studies showed increased SLPI with overexpressed NFX1-123 and co-expressed high-risk (HR) human papillomavirus type 16 E6 oncogene (16E6) in primary keratinocytes (HFKs) as well as high SPLI levels in cervical cancers. The HR viral oncogenes E6 and E7 partner with host cell proteins to regulate cell cycle regulation and immortalization pathways. Understanding the mechanisms of SLPI upregulation in HPV-associated cancers is critical to validating SLPI as a non-invasive biomarker as well as to test its utility in future cancer treatments. This study was designed to quantify intracellular and secreted SLPI protein in cervical cancer and to determine the roles NFX1-123 and 16E6 have on SLPI expression and secretion. Methods: Using HPV- and HPV+ cervical cancer cell lines and HFKs transduced with overexpressed NFX1-123 and 16E6, or vector controls, we measured intracellular and secreted SLPI by Western blot and ELISA, respectively. In HPV+ cervical cancer cell lines, NFX1-123 was knocked down or out by shRNA or CRISPR-Cas9, respectively, and 16E6 and 16E7 were knocked down by siRNA. In those cells, intracellular and secreted SLPI was quantified and the relative role of NFX1-123, 16E6, and 16E7 were studied. Selected classical and non-classical secretory pathways markers were also measured to assess their effects on SLPI secretion. Finally, SLPI’s concentration was measured by ELISA in cervical cancer patient sera and compared to healthy controls. Results: Our results showed increased intracellular as well as secreted SLPI in HFKs with overexpressed NFX1-123 and 16E6. Similarly, we found increased SLPI secretion in HPV+ cervical cancer cell lines (SiHa, Caski and HeLa) when compared to HPV- C33A cells. A reduction in NFX1-123 and 16E6 led to decreased SLPI secretion, pointing to a regulation of SLPI by NFX1-123 and 16E6 on SLPI secretion. Analysis of classical and non-classical secretory pathway markers suggested that NFX1-123 regulated non-classical pathways, which may drive greater secretion of SLPI in HPV+ cervical cancers. Higher SLPI protein amounts were found cervical cancer patient sera when compared to healthy controls. Conclusions: Our results suggest a functional role for NFX1-123 and 16E6 in SLPI secretion in cervical cancer and that SLPI may be a potential biomarker in cervical cancer. Citation Format: Sreenivasulu Chintala, Kevin Quist, Rachel Katzenellenbogen. Regulation of SLPI secretion by NFX1-123 and HPV 16 E6 in cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5919.

Abstract 1503: Augmenting genomic instability in HPV head and neck cancer tumors through combination bromodomain and extra-terminal (BET) proteins inhibition and radiation treatments

Abstract Integration of human papillomavirus (HPV)16 genomes into host head and neck squamous cell carcinoma (HNSCC) DNA is an essential driver for cancer progression. Currently, there are no treatment strategies based on HPV integration status. We postulate that a conceptual advance in the treatment of HPV HNSCC cancer can be formalized by targeting transcriptional co-regulator - bromodomain and extra-terminal (BET) proteins which mediate viral and host transcription. HPV maintenance in the host environment is dependent on BET protein-BRD4. An additional function of BET proteins in regulating transcription of tumor suppressors and DNA damage repair (DDR) genes. Given that patient populations have viral integrations into their genomes and its maintenance depends on cellular Brd4, displacing Brd4 through novel second-generation BET inhibitors can abrogate HPV transcription. Through chemical and knockdown studies of BET proteins in integrated viral HNSCC cells, we show viral oncogenes E6 and E7 are directly regulated by Brd4, reactivate tumor suppressors p53, release E2F1-Rb cell cycle brake to induce G1 cell cycle arrest, and significantly converge towards downregulation of DDR pathways in disrupted viral genomes. In contrast, non-disrupted viral genomes diverge from DDR pathways towards downregulation of interferon signaling pathways. As a result of the intrinsic genomic instability caused by the downregulation of components of the DDR machinery, the addition of external beam radiation further delayed DNA repair kinetics quantified through H2AX foci formation in cells harboring disrupted viral genomes. Taken together, targeting BET proteins is not a viable monotherapy but creates genomic instability, which, when combined with radiation, has the potential to eradicate HPV-associated tumors. Citation Format: Aakarsha Rao, Zijian Ni, Albert Wang, Dhruthi Suresh, Chitrasen Mohanty, Christina Kendziorski, Gopal Iyer. Augmenting genomic instability in HPV head and neck cancer tumors through combination bromodomain and extra-terminal (BET) proteins inhibition and radiation treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1503.

HPV E7 oncogene expression impairs Rb function and confers CDK4/6 inhibitor resistance in cervical cancer.

e17504 Background: Malignant tumor proliferation is one of the important factors for poor prognosis. CDK4/6 inhibitors can delay tumor progression by inhibiting cell cycle and inducing apoptosis, so they are potentially effective broad-spectrum anti-tumor targeted drugs, but drug resistance limits their clinical applications. Our previous research found that the CDK4/6 inhibitor LEE011 has anti-tumor effects on cervical cancer C33A cells, but no anti-tumor activity against HeLa. The mechanism is not very clear, we speculate that it is related to HPV infection. In this research, our aim is to clarify the correlation between HPV and tumor drug sensitivity and explore the possible mechanisms. Methods: Two HPV positive human cervical cancer cell lines, SiHa and Caski were chosen to verify the sensitivity to LEE011 by cell morphology, cell cycle and apoptosis. The antitumor activity of CDK4/6 inhibitor LEE011 by E7 knockin C33A and E7 knock-down HeLa cell lines was assessed by in vitro clonogenic assay, flow cytometry, and target inhibition verified by immunoblotting. Effects of LEE011 and E7 knock-down combination in vivo were studied by xenograft tumor regrowth delay, and tissue section immunohistochemistry. Results: SiHa and Caski were insensitive to LEE011 antitumor activity. Enhancement of drugsensitivity was lost in cell lines with HPV positive. After down-regulation of E7, LEE011 treatment increased the inhibition cell proliferation and pro-apoptosis of HeLa. Mechanistically, the loss of E7-induced Rb inactivation and LEE011 inhibited Rb phosphorylation, leading to cell-cycle arrest. In vivo, LEE011 inhibited tumor regrowth, with sustained inhibition of cyclin D-CDK4/6-Rb-E2F1 activity in E7 knock-down HeLa. However, up-regulation of E7 can reduce the ability of LEE011 to inhibit cell proliferation and pro-apoptosis of C33A. In summary, our study signifies inhibiting the CDK4/6 pathway by LEE011 in combination with down-regulation of E7 as a promising therapeutic strategy to treat cervical cancer. Conclusions: These findings suggested that HPV can affect Rb function by expressing oncoprotein E7 leading to resistance to targeted drug therapy, which means that HPV may be a biomarkers of drug therapy efficacy.

ATM Pathway Is Essential for HPV-Positive Human Cervical Cancer-Derived Cell Lines Viability and Proliferation.

Infection with some mucosal human papillomavirus (HPV) types is the etiological cause of cervical cancer and of a significant fraction of vaginal, vulvar, anal, penile, and head and neck carcinomas. DNA repair machinery is essential for both HPV replication and tumor cells survival suggesting that cellular DNA repair machinery may play a dual role in HPV biology and pathogenesis. Here, we silenced genes involved in DNA Repair pathways to identify genes that are essential for the survival of HPV-transformed cells. We identified that inhibition of the ATM/CHK2/BRCA1 axis selectively affects the proliferation of cervical cancer-derived cell lines, without altering normal primary human keratinocytes (PHK) growth. Silencing or chemical inhibition of ATM/CHK2 reduced the clonogenic and proliferative capacity of cervical cancer-derived cells. Using PHK transduced with HPV16 oncogenes we observed that the effect of ATM/CHK2 silencing depends on the expression of the oncogene E6 and on its ability to induce p53 degradation. Our results show that inhibition of components of the ATM/CHK2 signaling axis reduces p53-deficient cells proliferation potential, suggesting the existence of a synthetic lethal association between CHK2 and p53. Altogether, we present evidence that synthetic lethality using ATM/CHK2 inhibitors can be exploited to treat cervical cancer and other HPV-associated tumors.

Human papillomavirus and cervical cancer: an insight highlighting pathogenesis and targeting strategies.

Cervical cancer is marked by the uncontrolled proliferation and division of cells making up the cervix. Because of its enormous population, Asia accounts for more than half of the cervical cancer cases and deaths in the world. Cervical cancer is the major cause of death from cancer in women in rural as well as urban areas in India. In most cases, persistent infection with highly infectious types of human papillomavirus (HPV) such as HPV 16 and 18 is believed to be the cause of the disease. The HPV virus is primarily reported to invade cervical epithelial cells and then goes through a non-viremic infection cycle under the influence of various potent viral oncogenic proteins, namely E6 and E7. Among several other risk factors, increased oxidative stress, hyperactivation of inflammatory pathways, and immunological factors play a key role in cervical cancer pathogenesis. Although, standardized screening services in developed countries have substantially reduced the prevalence of cervical cancer but there are numerous drawbacks to cytology-based screening. Advances in understanding the virology of the human papillomavirus have prompted the discovery of several novel biomarkers of different categories such as protein-based, DNA-based as well as stem cell-based markers. The incorporation of biomarker information will assist in recognizing efficacious therapy systems as well as improve the prognosis of cervical malignancy. The review discussed the role of HPV in the development of cervical cancer and its pathogenesis. Further, summarized the potential therapeutic biomarkers for the prevention and treatment of cervical cancer.

Gene Expression Profile Analysis of Human Epidermal Keratinocytes Expressing Human Papillomavirus Type 8 E7.

Background: Human papillomavirus type 8 (HPV8) has been implicated in the progress of non-melanoma skin cancers and their precursor lesions. The HPV8 E7 oncoprotein plays a key role in the tumorigenesis of HPV-associated cutaneous tumors. However, the exact role of HPV8 E7 in human epidermal carcinogenesis has not been fully elucidated. Methods: To investigate the potential carcinogenic effects of HPV8 E7 on epithelial cells, we used RNA-sequencing technology to analyze the gene expression profile of HPV8 E7-overexpressed normal human epidermal keratinocytes (NHEKs). Results: RNA-sequencing revealed 831 differentially expressed genes (DEGs) between HPV8 E7-expressing NHEKs and control cells, among which, 631 genes were significantly upregulated, and 200 were downregulated. Gene ontology annotation enrichment analysis showed that HPV8 E7 mainly affected the expression of genes associated with protein heterodimerization activity, DNA binding, nucleosomes, and nucleosome assembly. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that overexpression of HPV8 E7 affected the expression of gene clusters associated with viral carcinogenesis and transcriptional misregulation in cancer and necroptosis signaling pathways that reportedly play crucial roles in HPV infection promotion and cancer progression. We also found the DEGs, such as HKDC1 and TNFAIP3, were associated with epigenetic modifications, immune regulation, and metabolic pathways. Conclusion: Our results demonstrate that the pro-carcinogenic effect of HPV8 expression in epithelial cells may be attributed to the regulatory effect of oncogene E7 on gene expression associated with epigenetic modifications and immune and metabolic status-associated gene expression. Although our data are based on an in vitro experiment, it provides the theoretical evidence that the development of squamous cell carcinoma can be caused by HPV.