Human Papillomavirus Oncoproteins Research Articles

HPV18 E7 inhibits LATS1 kinase and activates YAP1 by degrading PTPN14.

The Hippo kinase cascade inhibits YAP1, an oncoprotein and driver of cell stemness and self-renewal. There is mounting evidence that the Hippo pathway is targeted by tumor viruses including human papillomavirus. The high-risk HPV E7 oncoprotein promotes YAP1 nuclear localization and the carcinogenic activity of high-risk HPV E7 requires YAP1 activity. Blocking HPV E7-dependent YAP1 activation could inhibit HPV-mediated carcinogenesis, but the mechanism by which HPV E7 activates YAP1 has not been elucidated. Here we report that by degrading the tumor suppressor PTPN14, HPV18 E7 inhibits LATS1 kinase, reducing inhibitory phosphorylation on YAP1. These data support that an HPV oncoprotein can inhibit Hippo signaling to activate YAP1 and strengthen the link between PTPN14 and Hippo signaling in human epithelial cells.

High-risk HPV oncoproteins E6 and E7 and their interplay with the innate immune response: Uncovering mechanisms of immune evasion and therapeutic prospects.

Human papillomaviruses (HPVs) are double-stranded DNA (dsDNA) tumor viruses causally associated with 5% of human cancers, comprising both anogenital and upper aerodigestive tract carcinomas. Despite the availability of prophylactic vaccines, HPVs continue to pose a significant global health challenge, primarily due to inadequate vaccine access and coverage. These viruses can establish persistent infections by evading both the intrinsic defenses of infected tissues and the extrinsic defenses provided by professional innate immune cells. Crucial for their evasion strategies is their unique intraepithelial life cycle, which effectively shields them from host detection. Thus, strategies aimed at reactivating the innate immune response within infected or transformed epithelial cells, particularly through the production of type I interferons (IFNs) and lymphocyte-recruiting chemokines, are considered viable solutions to counteract the adverse effects of persistent infections by these oncogenic viruses. This review focuses on the complex interplay between the high-risk HPV oncoproteins E6 and E7 and the innate immune response in epithelial cells and HPV-associated cancers. In particular, it details the molecular mechanisms by which E6 and E7 modulate the innate immune response, highlighting significant progress in our comprehension of these processes. It also examines forward-looking strategies that exploit the innate immune system to ameliorate existing anticancer therapies, thereby providing crucial insights into future therapeutic developments.

Abstract 6082: Rapid on-site nucleic acid detection using CRISPR and digital signal processing for portable and integrated cervical cancer screening in low resource settings

Abstract Addressing the global disparity in cancer care necessitates the development of rapid and affordable nucleic acid (NA) testing technologies. This need is particularly critical for cervical cancer. Molecular detection of human papillomavirus (HPV) has emerged as a highly accurate screening method, surpassing traditional Pap smears. However, implementing this transition in low- and middle-income countries has been challenging due to the high costs and centralized facilities required for current NA tests. Here, we present CreDiT (CRISPR Enhanced Digital Testing), an advanced diagnostic system for rapid, on-site NA detection. CreDiT integrates two major technical breakthroughs: i) a one-pot CRISPR strategy that simultaneously amplifies both target NAs and analytical signals, and ii) a robust fluorescent detection method based on digital communication (encoding/decoding) technology. These innovations enhance CreDiT's practical utility, offering a rapid assay (<35 minutes) that integrates NA extraction and detection in a single streamlined workflow. Furthermore, CreDiT's straightforward probe design enables easy incorporation of new NA targets, while its compact device provides robust signal detection. We adapted CreDiT for point-of-care HPV screening by designing probes for high-risk HPV genes (HPV16, HPV18, HPV45, HPV31, HPV33, HPV58) and oncoprotein mRNAs (E6, E7, p16INK4a) and developing a portable CreDiT device capable of processing 12 samples. CreDiT demonstrated sensitive detection of cell-derived HPV DNA targets down to single copies and accurately identified HPV types in every clinical cervical brushing specimen (n = 121) we tested. This technology has the potential to facilitate prompt and reliable triaging of high-risk HPV, overcoming pathology bottlenecks and circumventing geographical and socioeconomic barriers to effective cervical cancer screening in resource-limited regions. This work has spearheaded recent screening research efforts in Uganda and Ghana. Citation Format: Chang Yeol Lee, Hyunho Kim, Hanna Lee, Thomas Randall, Amy Ly, Hakho Lee, Cesar M. Castro. Rapid on-site nucleic acid detection using CRISPR and digital signal processing for portable and integrated cervical cancer screening in low resource settings [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6082.

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

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. 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-β and vimentin) were significantly increased in BRCA1-deficient cells. Our results indicate that a deficiency of BRCA1 promotes the transactivation activity of HPV16E6E7 leading to increase of cell proliferation and invasion. HPV infection appears to have the potential to enhance the aggressiveness of breast cancers, especially those deficient in BRCA1.

PAD-mediated citrullination is a novel candidate diagnostic marker and druggable target for HPV-associated cervical cancer.

Citrullination is an emerging post-translational modification catalyzed by peptidyl-arginine deiminases (PADs) that convert peptidyl-arginine into peptidyl-citrulline. In humans, the PAD family consists of five isozymes (PADs 1-4, 6) involved in multiple diseases, including cancer. Given that high-risk (hr) human papillomaviruses (HPVs) are the etiological agents of cervical cancer, in this study, we sought to determine whether PAD-mediated protein citrullination would play a functional role in the HPV-driven transformation of epithelial cells. Here we show that both total protein citrullination and PAD4 expression levels are significantly associated with cervical cancer progression. Specifically, epithelial immunostaining for PAD4 revealed an increasingly higher histoscore from low-grade (CIN1) to high-grade (CIN2, CIN3) cervical intraepithelial neoplasia, and invasive squamous cell carcinoma (SCC) lesions, raising the attractive possibility that PAD4 may be used as tumor staging markers. Furthermore, taking advantage of the epidermoid cervical cancer cell line CaSki, which harbors multiple copies of the integrated HPV16 genome, we show that the expression of E6 and E7 HPV oncoproteins is impaired by treatment with the pharmacological pan-PAD inhibitor BB-Cl-amidine. Consistently, p53 and p21, two targets of HPV oncoproteins, are upregulated by the PAD inhibitor, which undergoes cell growth arrest and apoptosis. Altogether, these findings highlight a novel mechanism by which hrHPVs alter host regulatory pathways involved in cell cycle and survival to gain viral fitness, raising the possibility that PADs may represent an attractive target for developing novel host-targeting antivirals effective in preventing cervical cancer progression.

Bioinformatics analysis of photoexcited natural flavonoid glycosides as the inhibitors for oropharyngeal HPV oncoproteins

The presence of oropharyngeal human papillomavirus (HPV)-18 E6 and E7 oncoproteins is highly significant in the progression of oropharyngeal cancer. Natural flavonoid compounds have potential as photosensitizers for light-activated antimicrobial therapy against HPV-associated oropharyngeal cancer. This study evaluated five natural flavonoid glycosides including Fisetin, Kaempferol, Morin, Myricetin, and Quercetin as photosensitizers against HPV-18 E6 and E7 oncoproteins using computational methods. After obtaining the amino acid sequences of HPV-18 E6 and E7, various tools were used to predict and verify their properties. The PubChem database was then examined to identify potential natural flavonoid glycosides, followed by predictions of their drug-likeness and ADMET properties. Subsequently, molecular docking was conducted to enhance the screening accuracy and to gain insights into the interactions between the natural compounds and the active sites of HPV-18 E6 and E7 oncoproteins. The protein structures of E6 and E7 were predicted and validated to be reliable. The results of molecular docking demonstrated that Kaempferol exhibited the highest binding affinity to both E6 and E7. All compounds satisfied Lipinski's rules of drug-likeness, except Myricetin. They showed high absorption, distribution volume and similar ADMET profiles with no toxicity. In summary, natural flavonoid glycosides, especially Kaempferol, show potential as photosensitizers for antimicrobial photodynamic therapy against HPV-associated oropharyngeal cancer through inhibition of E6 and E7 oncoproteins. These findings provide insights into the development of novel therapeutic strategies based on antimicrobial photodynamic therapy.

Effect of HPV Oncoprotein on Carbohydrate and Lipid Metabolism in Tumor Cells.

High-risk HPV infection accounts for 99.7% of cervical cancer, over 90% of anal cancer, 50% of head and neck cancers, 40% of vulvar cancer, and some cases of vaginal and penile cancer, contributing to approximately 5% of cancers worldwide. The development of cancer is a complex, multi-step process characterized by dysregulation of signaling pathways and alterations in metabolic pathways. Extensive research has demonstrated that metabolic reprogramming plays a key role in the progression of various cancers, such as cervical, head and neck, bladder, and prostate cancers, providing the material and energy foundation for rapid proliferation and migration of cancer cells. Metabolic reprogramming of tumor cells allows for the rapid generation of ATP, aiding in meeting the high energy demands of HPV-related cancer cell proliferation. The interaction between Human Papillomavirus (HPV) and its associated cancers has become a recent focus of investigation. The impact of HPV on cellular metabolism has emerged as an emerging research topic. A significant body of research has shown that HPV influences relevant metabolic signaling pathways, leading to cellular metabolic alterations. Exploring the underlying mechanisms may facilitate the discovery of biomarkers for diagnosis and treatment of HPV-associated diseases. In this review, we introduced the molecular structure of HPV and its replication process, discussed the diseases associated with HPV infection, described the energy metabolism of normal cells, highlighted the metabolic features of tumor cells, and provided an overview of recent advances in potential therapeutic targets that act on cellular metabolism. We discussed the potential mechanisms underlying these changes. This article aims to elucidate the role of Human Papillomavirus (HPV) in reshaping cellular metabolism and the application of metabolic changes in the research of related diseases. Targeting cancer metabolism may serve as an effective strategy to support traditional cancer treatments, as metabolic reprogramming is crucial for malignant transformation in cancer.

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.

Mapping the landscape of HPV integration and characterising virus and host genome interactions in HPV-positive oropharyngeal squamous cell carcinoma.

Human papillomavirus (HPV) integration into the host genome is an important factor in HPV(+)OPSCC carcinogenesis, in conjunction with HPV oncoproteins E6/E7. However, a well-studied investigation about virus-host interaction still needs to be completed. Our objective is to characterise HPV integration to investigate potential mechanisms of tumourigenesis independent of E6/E7 oncoproteins. High-throughput viral integration detection was performed on 109 HPV(+)OPSCC tumours with relevant clinicopathological information. Of these tumours, 38 tumours underwent targeted gene sequencing, 29 underwent whole exome sequencing and 26 underwent RNA sequencing. HPV integration was detected in 94% of tumours (with a mean integration count of 337). Tumours occurring at the tonsil/oropharyngeal wall that exhibit higher PD-L1 expression demonstrated increased integration sites (p=.024). HPV exhibited a propensity for integration at genomic sites located within specific fragile sites (FRA19A) or genes associated with functional roles such as cell proliferation and differentiation (PTEN, AR), immune evasion (CD274) and glycoprotein biosynthesis process (FUT8). The viral oncogenes E2, E4, E6 and E7 tended to remain intact. HPV fragments displayed enrichment within host copy number variation (CNV) regions. However, insertions into genes related to altered homologous recombination repair were infrequent. Genes with integration had distinct expression levels. Fifty-nine genes whose expression level was affected by viral integration were identified, for example, EPHB1, which was reported to be involved in cellular protein metabolic process. HPV can promote oncogenesis through recurrent integration into functional host genome regions, leading to subsequent genomic aberrations and gene expression disruption. This study characterises viral integrations and virus-host interactions, enhancing our understanding of HPV-related carcinogenesis mechanisms.

Expression of Aberrant MicroRNAs and p16INK4a Associated with HPV (6, 11, 16, 18, 31, 33, 35, 42, 43, 44, 45, 52, 53, and 56) in Oral Dysplasia and Squamous Cell Carcinoma: A Retrospective Study.

A few studies indicate that human papillomavirus (HPV) induces aberrant expression of microRNAs (miRNAs) and correlate this with p16INK4a in oral dysplasia (OD) and oral squamous cell carcinoma (OSCC). Therefore, this study aimed to evaluate the expression of miRNA-21, miRNA-22, and miRNA-224 by q-PCR and the p16 INK4a by immunohistochemical (IHC) as markers for HPV-positive OSCC and OD in comparison to controls as miRNA expression can be altered by the HPV oncogenes and hence can be used as a biomarker for HPV positive cases. Fifty-two specimens were collected from archived paraffin blocks for patients aged between 19 and 88 (31 males and 21 females) from various oral sites. They were examined by IHC using p16 INK4a, by RT-PCR for the detection of HPV (6, 11, 16, 18, 31, 33, 35, 42, 43, 44, 45, 52, 53, 56), and by q-PCR for the expression of miRNA-21, miRNA-22, and miRNA-224 in positive specimens. Out of the 15 OD, three were positive by both techniques. Meanwhile, 17 out of all OSCC specimens showed intense nuclear and cytoplasmic staining by p16 INK4a, and only 16 were also positive by RT-PCR. However, all control specimens were negative. MiRNA-21, miRNA-22, and miRNA-224 were overexpressed in 3 specimens of OD and 16 of OSCC. MiRNA-21, miRNA-22, and miRNA-224, besides p16 INK4a, could be used as indicators for HPV-associated OD and OSCC as their expression is attributed to the HPV oncoprotein. Further studies using follow-up data should be done to correlate it with miRNA overexpression.

Computational and AI-driven 3D structural analysis of human papillomavirus (HPV) oncoproteins E5, E6, and E7 reveal significant divergence of HPV E5 between low-risk and high-risk genotypes

There are over 220 identified genotypes of Human papillomavirus (HPV), and the HPV genome encodes 3 major oncogenes, E5, E6, and E7. Conservation and divergence in protein sequence and function between low-risk versus high-risk oncogenic HPV genotypes has not been fully characterized. Here, we used modern computational and structural folding algorithms to perform a comparative analysis of HPV E5, E6, and E7 between multiple low risk and high risk genotypes. We first identified significantly greater sequence divergence in E5 between low- and high-risk genotypes compared to E6 and E7. Next, we used AlphaFold to model the structure of papillomavirus proteins and complexes with high confidence, including some with no established consensus structure. We observed that HPV E5, but not E6 or E7, had a dramatically different 3D structure between low-risk and high-risk genotypes. To our knowledge, this is the first comparative analysis of HPV proteins using Alphafold artificial intelligence (AI) system. The marked differences in E5 sequence and structure in high-risk HPVs may contribute in important and underappreciated ways to the development of HPV-associated cancers.

The effects of HPV oncoproteins on host communication networks: Therapeutic connotations.

Human papillomavirus (HPV) infections are a leading cause of viral-induced malignancies worldwide, with a prominent association with cervical and head and neck cancers. The pivotal role of HPV oncoproteins, E5, E6, and E7, in manipulating cellular events, which contribute to viral pathogenesis in various ways, has been extensively documented. This article reviews the influence of HPV oncoproteins on cellular signaling pathways within the host cell, shedding light on the underlying molecular mechanisms. A comprehensive understanding of these molecular alterations is essential for the development of targeted therapies and strategies to combat HPV-induced premalignancies and prevent their progress to cancer. Furthermore, this review underscores the intricate interplay between HPV oncoproteins and some of the most important cellular signaling pathways: Notch, Wnt/β-catenin, MAPK, JAK/STAT, and PI3K AKT/mTOR. The treatment efficacies of the currently available inhibitors on these pathways in an HPV-positive context are also discussed. This review also highlights the importance of continued research to advance our knowledge and enhance therapeutic interventions for HPV-associated diseases.

9-oxo-ODAs suppresses the proliferation of human cervical cancer cells through the inhibition of CDKs and HPV oncoproteins

Mucosal human papillomavirus (HPV) subtypes 16 and 18 are causative agents of cervical cancer, a leading cause of cancer-related deaths among women worldwide. In Japan, eggplant calyx is a folk remedy used to treat common warts. 9-oxo-(10E,12E)-octadecadienoic acid, isolated from eggplant calyx, may have antitumor effects. This study investigated the antitumor effects of 9-oxo-(10E, 12Z)-octadecadienoic acid and 9-oxo-(10E,12E)-octadecadienoic acid (9-oxo-ODAs) on human cervical cancer cells. 9-oxo-ODAs suppressed the proliferation of human cervical cancer cell lines (HeLa, and SiHa) in a concentration-dependent manner (IC50 = 25–50 µM). FCM analysis revealed that 9-oxo-ODAs induced apoptosis. Transcriptome, proteomics, and enrichment analyses revealed that treatment with 9-oxo-ODAs significantly altered the cell cycle and p53 pathways and decreased cyclin-dependent kinase 1 (CDK1) protein expression. Real-time PCR analysis demonstrated that 9-oxo-ODAs reduced CDK1 mRNA expression in a concentration-dependent manner. In vitro, 9-oxo-ODAs reduced the HPV oncoprotein expression. In ex vivo human cervical cancer tissues, 9-oxo-ODAs decreased CDK1 expression and increased cleaved caspase 3, an apoptosis marker. Further, 9-oxo-ODAs showed the potential to suppressed metastatic formation and growth of cervical cancer in vivo. These findings suggest that 9-oxo-ODAs induce cell cycle arrest and apoptosis in HPV-positive human cervical cancer cells, and this process involves CDK1. Consequently, 9-oxo-ODAs may be potential therapeutic agents for cervical cancer.

C-MYC-dependent transcriptional inhibition of autophagy is implicated in cisplatin sensitivity in HPV-positive head and neck cancer

Autophagy is important for the removal, degradation and recycling of damaged organelles, proteins, and lipids through the degradative action of lysosomes. In addition to its catabolic function, autophagy is important in cancer and viral-mediated tumorigenesis, including Human Papillomavirus (HPV) positive cancers. HPV infection is a major risk factor in a subset of head and neck cancer (HNC), for which no targeted therapies are currently available. Herein, we assessed autophagy function in HPV-positive HNC. We showed that HPV-positive HNC cells presented a transcriptional and functional impairment of the autophagic process compared to HPV-negative cells, which were reactivated by knocking down HPV E6/E7 oncoproteins, the drivers of cellular transformation. We found that the oncoprotein c-MYC was stabilized and triggered in HPV-positive cell lines. This resulted in the reduced binding of the MiT/TFE transcription factors to their autophagy targets due to c-MYC competition. Thus, the knock-down of c-MYC induced the upregulation of autophagic and lysosomal genes in HPV-positive HNC cells, as well as the increase of autophagic markers at the protein level. Moreover, HPV oncoprotein E7 upregulated the expression of the phosphatase inhibitor CIP2A, accounting for c-MYC upregulation and stability in HPV+ HNC cells. CIP2A mRNA expression negatively correlated with autophagy gene expression in tumor tissues from HNC patients, showing, for the first time, its implication in a transcriptional autophagic context. Both CIP2A and c-MYC knock-down, as well as pharmacological downregulation of c-MYC, resulted in increased resistance to cisplatin treatment. Our results not only show a novel way by which HPV oncoproteins manipulate the host machinery but also provide more insights into the role of autophagy in chemoresistance, with possible implications for targeted HPV-positive HNC therapy.

Silencing E6/E7 Oncoproteins in SiHa Cells Treated with siRNAs and Oroxylum indicum Extracts Induced Apoptosis by Upregulating p53/pRb Pathways.

E6 and E7 human papillomavirus (HPV) oncoproteins play a significant role in the malignant transformation of infected cervical cancer cells via suppression of tumour suppressor pathways by targeting p53 and pRb, respectively. This study aimed to investigate the anticancer effects of Oroxylum indicum (OI) leaves' methanol extract on SiHa cervical cancer cells. Expression of apoptosis-related proteins (Bcl-2, caspase (cas)-3, and cas-9), viral oncoproteins (E6 and E7), and tumour suppressor proteins (p53 and pRb) were evaluated using western blot analysis before and after E6/E7 small interfering RNAs (siRNAs) transfection. In addition, the E6/E7 mRNA expression levels were assessed with real-time (RT)-PCR. The present study showed that the OI extract effectively hindered the proliferation of SiHa cells and instigated increments of cas-3 and cas-9 expressions but decreased the Bcl-2 expressions. The OI extract inhibited E6/E7 viral oncoproteins, leading to upregulation of p53 and pRb tumour suppressor genes in SiHa cells. Additionally, combinatorial treatment of OI extract and gossypin flavonoid induced restorations of p53 and pRb. Treatment with OI extract in siRNA-transfected cells also further suppressed E6/E7 expression levels and further upregulations of p53 and pRb proteins. In conclusion, OI extract treatment on siRNAs-transfected SiHa cells can additively and effectively block E6- and E7-dependent p53 and pRb degradations. All these data suggest that OI could be explored for its chemotherapeutic potential in cervical cancer cells with HPV-integrated genomes.

Recent Updates on the Management of Human Papillomavirus-related Cancers

Abstract: Cervical cancer incidence and mortality rates have been steadily decreasing in developed nations owing to the excellent screening programs executed. However, it still remains one of the most prevalent tumors in developing nations, contributing significantly to cancer-related mortality in females. The major causal factor in the genesis of cervical cancer is recognized to be human papillomavirus (HPV) infection. The female population, particularly in poor countries, is highly susceptible to HPV infections and cervical cancer as a result of the increasing costs posed by widespread cervical screening and HPV vaccination methods. Understanding the roles of HPV oncoproteins (E6 and E7) and non-coding RNAs, along with their many cellular targets, can help us develop targeted drug therapy to manage cervical cancer. In the hunt for novel ways for effective disease control and prevention, new insights and methodologies in molecular biology keep evolving continuously. In the recent past, newer studies have revealed deeper knowledge of HPV-activated molecular signaling pathways as well as prospective targets for early diagnosis, prevention, and therapy of HPV-related malignancies. Also, there has been much new research conducted on genome-editing tools for HPVinduced cervical cancer treatment in conjunction with other treatment strategies, such as immunotherapy and therapeutic vaccines.

Abstract PR04: Deciphering radiotherapy resistance mechanisms in HPV negative and positive head and neck cancers

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. The prognosis of HNSCC is poor with a 5-year survival rate for all stages around 55%. HNSCC can be classified into two subtypes, human papilloma virus (HPV)-negative, caused mainly by tobacco use, and HPV-positive subtype. Radiotherapy is one of the cornerstones of the treatment of HNSCC. However, radioresistance is common and is associated with a high risk of recurrence. Radioresistance may be pre-existing, or acquired, and may be direct (tumor-cell autonomous) or indirect, mediated by RT-mediated changes to the TME such as cellular and/or extracellular matrix (ECM) changes to the stroma. These pathways are not fully understood. Better understanding of the intrinsic as well as RT-induced resistance is needed to improve survival of hard-to-treat HNSCC patients. HPV-positive HNSCC have significantly better RT response and prognosis, however currently they are treated with similarly high dose RT which causes significant adverse side-effects. As the incidence of HPV-induced HNSCC is increasing very fast, the current focus is on treatment de-escalation to reduce toxicity without compromising outcome. EGFR upregulation is an established biomarker of treatment resistance and aggressiveness in head HNSCC. EGFR-targeted therapies have shown benefits for HPV-negative HNSCC; surprisingly, inhibiting EGFR in HPV-associated HNSCC led to inferior therapeutic outcomes suggesting opposing roles for EGFR in the two HNSCC subtypes. We have recently shown that EGFR activation regulates the DNA damage response pathway differently in the two HNSCC subtype. Here, we further investigated the link between EGFR with HPV-infected HNSCC particularly the regulation of HPV oncoproteins E6 and E7. We demonstrate that EGFR overexpression suppresses cellular proliferation and increases radiosensitivity of HPV-positive HNSCC cell lines. EGFR overexpression was shown to inhibit protein expression of BRD4, a known cellular transcriptional regulator of HPV E6/E7 expression and DNA damage repair facilitator. Using in vitro 2D and 3D spheroid models of HNSCC we showed that EGFR inhibition by cetuximab restored the expression of BRD4 leading to increased HPV E6 and E7 transcription. Concordantly, pharmacological inhibition of BRD4 led to suppression of HPV E6 and E7 transcription, delayed cellular proliferation and sensitised HPV-positive HNSCC cells to ionising radiation. This effect was shown to be mediated through EGFR-induced upregulation of microRNA-9-5p and consequent silencing of its target BRD4 at protein translational level, repressing HPV E6 and E7 transcription and restoring p53 tumor suppressor functions. These results suggest a novel mechanism for EGFR inhibition of HPV E6/E7 oncoprotein expression through an epigenetic pathway, mediated through microRNA-9-5p/BRD4 regulation. The results may help better future design of radiotherapy combination treatment of HPV-positive and negative HNSCC, such as combination with BRD4 inhibitors to improve HNSCC therapeutic outcome. Citation Format: Mahvash Tavassoli. Deciphering radiotherapy resistance mechanisms in HPV negative and positive head and neck cancers [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PR04.

CREB1 activation promotes human papillomavirus oncogene expression and cervical cancer cell transformation.

Human papillomaviruses (HPVs) infect the oral and anogenital mucosa and can cause cancer. The high-risk (HR)-HPV oncoproteins, E6 and E7, hijack cellular factors to promote cell proliferation, delay differentiation and induce genomic instability, thus predisposing infected cells to malignant transformation. cAMP response element (CRE)-binding protein 1 (CREB1) is a master transcription factor that can function as a proto-oncogene, the abnormal activity of which is associated with multiple cancers. However, little is known about the interplay between HPV and CREB1 activity in cervical cancer or the productive HPV lifecycle. We show that CREB is activated in productively infected primary keratinocytes and that CREB1 expression and phosphorylation is associated with the progression of HPV+ cervical disease. The depletion of CREB1 or inhibition of CREB1 activity results in decreased cell proliferation and reduced expression of markers of epithelial to mesenchymal transition, coupled with reduced migration in HPV+ cervical cancer cell lines. CREB1 expression is negatively regulated by the tumor suppressor microRNA, miR-203a, and CREB1 phosphorylation is controlled through the MAPK/MSK pathway. Crucially, CREB1 directly binds the viral promoter to upregulate transcription of the E6/E7 oncogenes, establishing a positive feedback loop between the HPV oncoproteins and CREB1. Our findings demonstrate the oncogenic function of CREB1 in HPV+ cervical cancer and its relationship with the HPV oncogenes.

Modern concepts in cervical carcinogenesis

The article discusses modern ideas about cervical carcinogenesis as a multi-stage process of multifactorial genesis. Currently, ideas about the pathogenesis of cervical cancer (CC) are based not only on understanding the role of high-risk oncogenic human papillomavirus (HPV) in this process and accumulation of genetic changes caused by it, but also on formation of a complex HPV interactome, or a network of intermolecular interactions of HPV oncoproteins with host cell proteins. Carcinogenesis also involves a wide range of epigenetic events and, above all, impairment of the regulatory function of miRNAs. An important role in cervical carcinogenesis is attributed to the concept of cancer stem cells (CSCs) formulated in recent years, which is closely related to the explanation of disease recurrence and treatment resistance, as well as to new approaches to treatment. The cervicovaginal microbiome and cervical microenvironment, which are responsible for natural clearance of HPV, regression of epithelial lesions, and modeling of the immune response, are becoming promising objects for research.The aim of the review was to present up-to-date information on the most important mechanisms of cervical carcinogenesis, as well as on new approaches to the treatment of CC, based, in particular, on the use of knowledge about regulatory miRNAs, CSC markers, and the state of the cervicovaginal microbiota.

The Tumor-Specific Immune Landscape in HPV+ Head and Neck Cancer.

Human papillomaviruses (HPVs) are the causative agent of several anogenital cancers as well as head and neck cancers, with HPV+ head and neck squamous cell carcinoma (HNSCC) becoming a rapidly growing public health issue in the Western world. Due its viral etiology and potentially its subanatomical location, HPV+ HNSCC exhibits an immune microenvironment which is more inflamed and thus distinct from HPV-negative HNSCC. Notably, the antigenic landscape in most HPV+ HNSCC tumors extends beyond the classical HPV oncoproteins E6/7 and is extensively targeted by both the humoral and cellular arms of the adaptive immune system. Here, we provide a comprehensive overview of HPV-specific immune responses in patients with HPV+ HNSCC. We highlight the localization, antigen specificity, and differentiation states of humoral and cellular immune responses, and discuss their similarities and differences. Finally, we review currently pursued immunotherapeutic treatment modalities that attempt to harness HPV-specific immune responses for improving clinical outcomes in patients with HPV+ HNSCC.