Human Papillomavirus E6 Oncoproteins Research Articles

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.

MSphere of Influence: Retreading old ground-the beauty in multi-omic data sets.

Ethan L. Morgan works on human papillomaviruses (HPVs), with a specific interest in identifying how HPV induces tumor formation. In this mSphere of Influence article, he reflects on how three papers influenced him. "Comprehensive genomic characterization of head and neck squamous cell carcinomas" (The Cancer Genome Atlas Network, Nature 517:576-582, 2015, https://doi.org/10.1038/nature14129) and "Integrated genomic and molecular characterization of cervical cancer" (The Cancer Genome Atlas Network, Nature 543: 378-384, 2017, https://doi.org/10.1038/nature21386) showed him the power behind comprehensive multi-omic analyses to understand disease biology, while "Human papillomavirus E7 oncoprotein targets RNF168 to hijack the host DNA damage response" (J. Sitz et al., Proc Natl Acad Sci U S A 116:19552-19562, 2019, https://doi.org/10.1073/pnas.1906102116) reinforced how this can be used to undercover potential new drug targets in HPV-associated disease.

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.

Revolutionizing HPV Detection: Unveiling a Novel Electrochemical Immunosensor for Ultra-Sensitive HPV16 E7 Oncoprotein Detection Based on Gold-Graphitic Carbon Nitride Nanocomposite

Cervical cancer (CC), a significant contributor to the mortality of females globally, manifests an annual incidence of 500,000 cases and is predominantly linked to the human papillomavirus (HPV) infection. The pressing demand for affordable and user-friendly assays for CC detection is highlighted by the limited availability and dependability of diagnostic screening tools, especially in developing countries. In response to the critical need for enhanced cervical cancer diagnostics, we present an innovative electrochemical immunosensor designed for the selective detection of the HPV 16-E7 oncoprotein. The immunosensing platform is constructed from a gold-graphitic carbon nitride (Au-g-C3N4) nanocomposite, with antibodies immobilized through a physical adsorption process onto the sensing platform. Employing Square Wave Voltammetry (SWV), we systematically evaluated the antigen-antibody interaction, achieving ultrasensitive detection of E7 with ferric/ferrocyanide. The linear detection range of the proposed sensor for E7 antigen is 0.01 fg ml−1 to 1 ng ml−1, assuming ideal conditions and an impressive Limit of Detection (LOD) of 0.0065 fg ml−1. The accuracy of the proposed immunosensor was effectively evaluated in contrast to Enzyme linked immunosorbent assay (ELISA). Finally, the proposed immunosensor exhibited favourable selectivity, sensitivity, and reproducibility upon introduction to clinical samples, thereby validating its potential suitability for point-of-care testing aimed at the timely detection of CC.

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.

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.

Comparative Analysis of Alpha and Beta HPV E6 Oncoproteins: Insights into Functional Distinctions and Divergent Mechanisms of Pathogenesis.

Human papillomaviruses (HPVs) represent a diverse group of DNA viruses that infect epithelial cells of mucosal and cutaneous tissues, leading to a wide spectrum of clinical outcomes. Among various HPVs, alpha (α) and beta (β) types have garnered significant attention due to their associations with human health. α-HPVs are primarily linked to infections of the mucosa, with high-risk subtypes, such as HPV16 and HPV18, being the major etiological agents of cervical and oropharyngeal cancers. In contrast, β-HPVs are predominantly associated with cutaneous infections and are commonly found on healthy skin. However, certain β-types, notably HPV5 and HPV8, have been implicated in the development of non-melanoma skin cancers in immunocompromised individuals, highlighting their potential role in pathogenicity. In this review, we comprehensively analyze the similarities and differences between α- and β-HPV E6 oncoproteins, one of the major drivers of viral replication and cellular transformation, and how these impact viral fitness and the capacity to induce malignancy. In particular, we compare the mechanisms these oncoproteins use to modulate common cellular processes-apoptosis, DNA damage repair, cell differentiation, and the immune response-further shedding light on their shared and distinct features, which enable them to replicate at divergent locations of the human body and cause different types of 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.

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.

Increased expression of HPV-E7 oncoprotein correlates with a reduced level of pRb proteins via high viral load in cervical cancer

Human Papillomavirus (HPV) is the most common cause of sexually transmitted diseases and causes a wide range of pathologies including cervical carcinoma. Integration of the HR-HPV DNA into the host genome plays a crucial role in cervical carcinoma. An alteration of the pRb pathways by the E7 proteins is one of the mechanisms that’s account for the transforming capacity of high-risk papillomavirus. For the proper understanding of the underline mechanism of the progression of the disease, the present study investigate the correlation of concentration of host pRb protein, viral E7 oncoprotein and viral load in early and advanced stages of cervical carcinoma. It was found that the viral load in early stages (stage I and II) was less (log10 transformed mean value 2.6 and 3.0) compared to advanced stages (stage III and IV) (Log10 transformed value 5.0 and 5.8) having high expression of HPV E7 onco-protein and reduced level of pRb protein, signifying the role of viral load and expression level of E7 oncoprotein in the progression of cervical cancer.

Expression of HPV-16 E6 and E7 oncoproteins alters Chlamydia trachomatis developmental cycle and induces increased levels of immune regulatory molecules.

Infection with Human Papillomavirus (HPV) is a recognized risk factor for Chlamydia trachomatis (CT) infection and vice versa. Coinfection of HPV and CT in women is a very common and usually asymptomatic finding that has been linked to increased risk of cervical cancer. It has been demonstrated that CT facilitates the entry of multiple high risk HPV genotypes, leading to damage of the mucosal barrier and interfering with immune responses and viral clearance, which ultimately favours viral persistence and malignant transformation. Although the facilitating effects elicited by CT infection on viral persistence have been reported, little is known about the consequences of HPV infection on CT development. Herein, we took advantage of a genetically modified human cervical cell line co-expressing HPV-16 major oncogenic proteins E6 and E7, as an experimental model allowing to investigate the possible effects that HPV infection would have on CT development. Our results show that CT infection of HPV-16 E6E7 expressing cells induced an upregulation of the expression of E6E7 oncoproteins and host cell inhibitory molecules PD-L1, HVEM and CD160. Additionally, smaller chlamydial inclusions and reduced infectious progeny generation was observed in E6E7 cells. Ultrastructural analysis showed that expression of E6 and E7 did not alter total bacterial counts within inclusions but resulted in increased numbers of reticulate bodies (RB) and decreased production of infectious elementary bodies (EB). Our results indicate that during CT and HPV coinfection, E6 and E7 oncoproteins impair RB to EB transition and infectious progeny generation. On the other hand, higher expression of immune inhibitory molecules and HPV-16 E6E7 are cooperatively enhanced in CT-infected cells, which would favour both oncogenesis and immunosuppression. Our findings pose important implications for clinical management of patients with HPV and CT coinfection, suggesting that screening for the mutual infection could represent an opportunity to intervene and prevent severe reproductive health outcomes, such as cervical cancer and infertility.

A small molecule targeting the interaction between human papillomavirus E7 oncoprotein and cellular phosphatase PTPN14 exerts antitumoral activity in cervical cancer cells

Human papillomavirus (HPV)-induced cancers still represent a major health issue for worldwide population and lack specific therapeutic regimens. Despite substantial advancements in anti-HPV vaccination, the incidence of HPV-related cancers remains high, thus there is an urgent need for specific anti-HPV drugs. The HPV E7 oncoprotein is a major driver of carcinogenesis that acts by inducing the degradation of several host factors. A target is represented by the cellular phosphatase PTPN14 and its E7-mediated degradation was shown to be crucial in HPV oncogenesis. Here, by exploiting the crystal structure of E7 bound to PTPN14, we performed an in silico screening of small-molecule compounds targeting the C-terminal CR3 domain of E7 involved in the interaction with PTPN14. We discovered a compound able to inhibit the E7/PTPN14 interaction in vitro and to rescue PTPN14 levels in cells, leading to a reduction in viability, proliferation, migration, and cancer-stem cell potential of HPV-positive cervical cancer cells. Mechanistically, as a consequence of PTPN14 rescue, treatment of cancer cells with this compound altered the Yes-associated protein (YAP) nuclear-cytoplasmic shuttling and downstream signaling. Notably, this compound was active against cervical cancer cells transformed by different high-risk (HR)-HPV genotypes indicating a potential broad-spectrum activity. Overall, our study reports the first-in-class inhibitor of E7/PTPN14 interaction and provides the proof-of-principle that pharmacological inhibition of this interaction by small-molecule compounds could be a feasible therapeutic strategy for the development of novel antitumoral drugs specific for HPV-associated cancers.

Does Human Papillomavirus Play a Causative Role in Prostate Cancer? A Systematic Review Using Bradford Hill's Criteria.

Globally, prostate cancer is the fifth most common cause of cancer-related death among men, and metastatic castration-resistant prostate cancer has a high cancer-related mortality rate. However, the aetiology of this disease is not yet fully understood. While human papillomavirus (HPV) has been associated with several types of cancer, including cervical, anal, and oropharyngeal cancers, studies investigating the relationship between HPV and prostate cancer have shown mixed results. This systematic review aimed to evaluate the causative association between HPV and prostate cancer using Bradford Hill's criteria. A comprehensive search of PubMed was conducted, and 60 out of 482 studies were included in the review. The included studies were evaluated based on nine Bradford Hill criteria, and information on the identification and transmission of the virus and potential oncogenic mechanisms was also extracted. The strength of association criterion was not met, and other criteria, such as consistency and coherence, were not fulfilled. However, biological plausibility was supported, and potential oncogenic mechanisms were identified. While some studies have reported the presence of HPV in prostate cancer tissues, the overall quality of evidence remains low, and the association between HPV and prostate cancer is weak. Nevertheless, the prostate is a potential reservoir for the transmission of HPV, and the HPV E6 and E7 oncoproteins and inflammation are likely to be involved in any oncogenic mechanisms. Further studies with a higher level of evidence are needed to establish a definitive link between HPV and prostate cancer.

HPV18 E6 inhibits α-ketoglutarate-induced pyroptosis of esophageal squamous cell carcinoma cells via the P53/MDH1/ROS/GSDMC pathway.

Oncogene E6 plays a critical role in the development and progression of esophageal cancer caused by human papillomavirus (HPV) infection. Alpha-ketoglutarate (AKG) is a key metabolite in the tricarboxylic acid cycle and has been widely used as a dietary and anti-ageing supplement. In this study, we found that treating esophageal squamous carcinoma cells with a high dose of AKG can induce cell pyroptosis. Furthermore, our research confirms that HPV18 E6 inhibits AKG-induced pyroptosis of esophageal squamous carcinoma cells by lowering P53 expression. P53 downregulates malate dehydrogenase 1 (MDH1) expression; however, MDH1 downregulates L-2-hydroxyglutarate (L-2HG) expression, which inhibits a rise in reactive oxygen species (ROS) levels-as L-2HG is responsible for excessive ROS. This study reveals the actuating mechanism behind cell pyroptosis of esophageal squamous carcinoma cells induced by high concentrations of AKG, and we posit the molecular pathway via which the HPV E6 oncoprotein inhibits cell pyroptosis.

JAK/STAT Signaling and Cervical Cancer: From the Cell Surface to the Nucleus.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway constitutes a rapid signaling module from the cell surface to the nucleus, and activates different cellular responses, such as proliferation, survival, migration, invasion, and inflammation. When the JAK/STAT pathway is altered, it contributes to cancer progression and metastasis. STAT proteins play a central role in developing cervical cancer, and inhibiting the JAK/STAT signaling may be necessary to induce tumor cell death. Several cancers show continuous activation of different STATs, including cervical cancer. The constitutive activation of STAT proteins is associated with a poor prognosis and overall survival. The human papillomavirus (HPV) oncoproteins E6 and E7 play an essential role in cervical cancer progression, and they activate the JAK/STAT pathway and other signals that induce proliferation, survival, and migration of cancer cells. Moreover, there is a crosstalk between the JAK/STAT signaling cascade with other signaling pathways, where a plethora of different proteins activate to induce gene transcription and cell responses that contribute to tumor growth. Therefore, inhibition of the JAK/STAT pathway shows promise as a new target in cancer treatment. In this review, we discuss the role of the JAK/STAT pathway components and the role of the HPV oncoproteins associated with cellular malignancy through the JAK/STAT proteins and other signaling pathways to induce tumor growth.

Activation of p53-regulated pro-survival signals and hypoxia-independent mitochondrial targeting of TIGAR by human papillomavirus E6 oncoproteins

The high-risk subtype human papillomaviruses (hrHPVs) infect and oncogenically transform basal epidermal stem cells associated with the development of squamous-cell epithelial cancers. The viral E6 oncoprotein destabilizes the p53 tumor suppressor, inhibits p53 K120-acetylation by the Tat-interacting protein of 60 kDa (TIP60, or Kat5), and prevents p53-dependent apoptosis. Intriguingly, the p53 gene is infrequently mutated in HPV + cervical cancer clinical isolates which suggests a possible paradoxical role for this gatekeeper in viral carcinogenesis. Here, we demonstrate that E6 activates the TP53-induced glycolysis and apoptosis regulator (TIGAR) and protects cells against oncogene-induced oxidative genotoxicity. The E6 oncoprotein induces a Warburg-like stress response and activates PI3K/PI5P4K/AKT-signaling that phosphorylates the TIGAR on serine residues and induces its hypoxia-independent mitochondrial targeting in hrHPV-transformed cells. Primary HPV + cervical cancer tissues contain high levels of TIGAR, p53, and c-Myc and our xenograft studies have further shown that lentiviral-siRNA-knockdown of TIGAR expression inhibits hrHPV-induced tumorigenesis in vivo. These findings suggest the modulation of p53 pro-survival signals and the antioxidant functions of TIGAR could have key ancillary roles during HPV carcinogenesis.

MRNA-HPV Therapeutic Vaccine Encoding E6 and E7 Improves Therapeutic Potential in HPV-mediated Cancer

Abstract The viral E6 and E7 oncoproteins of human papillomavirus (HPV) are strongly associated with cervical cancer. We developed and assessed the efficacy of an mRNA-HPV therapeutic vaccine (mHTV), containing the E6 and E7 proteins, in C57BL/J mice. mHTV generated robust T cell-mediated immune responses and induced short-lived effector and E7 tetramer-specific T cells. mHTV immunization significantly suppressed tumor growth in mice implanted with TC-1 tumor cells and improved overall survival. The re-transplantation of tumor cells into mice that survived the primary tumor challenge after primary vaccination led to the complete inhibition of tumor progression in all mHTV-treated mice. This showed that mHTV can effectively suppress cancer recurrence. mHTV also increased the number of tumor-infiltrating lymphocytes in the tumor tissues. When mice were implanted with TC-1 tumor cells after mHTV vaccination, most of the immunized mice were protected from the tumor challenge. In conclusion, mHTV showed considerable potential as not only a therapeutic vaccine, by efficiently killing cancer cells, but also as a prophylactic vaccine by enhancing T cell-mediated immune responses. The Brain Korea 21 Plus Program, Partially supported. The Ministry of Food and Drug Safety grant (grant number 22213MFDS421) The National Research Foundation of Korea (NRF) grant (grant number NRF-2021M3E5E3080558)

Interaction between Human Papillomavirus-Encoded E6 Protein and AurB Induces Cell Immortalization and Proliferation-A Potential Target of Intervention.

The human papillomavirus E6 and E7 oncoproteins interact with a different subset of host proteins, leading to dysregulation of the apoptotic, cell cycle, and signaling pathways. In this study, we identified, for the first time, that Aurora kinase B (AurB) is a bona fide interacting partner of E6. We systematically characterized the AurB-E6 complex formation and its consequences in carcinogenesis using a series of in vitro and cell-based assays. We also assessed the efficacy of Aurora kinase inhibitors in halting HPV-mediated carcinogenesis using in vitro and in vivo models. We showed that AurB activity was elevated in HPV-positive cells, and this correlated positively with the E6 protein level. E6 interacted directly with AurB in the nucleus or mitotic cells. A previously unidentified region of E6, located upstream of C-terminal E6-PBM, was important for AurB-E6 complex formation. AurB-E6 complex led to reduced AurB kinase activity. However, the AurB-E6 complex increased the hTERT protein level and its telomerase activity. On the other hand, AurB inhibition led to the inhibition of telomerase activity, cell proliferation, and tumor formation, even though this may occur in an HPV-independent manner. In summary, this study dissected the molecular mechanism of how E6 recruits AurB to induce cell immortalization and proliferation, leading to the eventual cancer development. Our findings revealed that the treatment of AZD1152 exerted a non-specific anti-tumor effect. Hence, a continuous effort to seek a specific and selective inhibitor that can halt HPV-mediated carcinogenesis should be warranted.

Microwave hyperthermia represses human papillomavirus oncoprotein activity and induces cell death due to cell stress in 3D tissue models of anogenital precancers and cancers

Hyperthermia is a well-accepted cancer therapy. Microwaves provide a very precise, targeted means of hyperthermia and are currently used to treat plantar warts caused by cutaneous-infective human papillomaviruses (HPVs). Other HPV genotypes infecting the anogenital mucosa cause genital warts or preneoplastic lesions or cervical cancer. Effective, non-ablative therapies for these morbid HPV-associated lesions are lacking. The molecular consequences of microwave treatment were investigated in invitro cultured three-dimensional HPV-positive cervical tumour tissues, and tissues formed from HPV-infected normal immortalised keratinocytes. Microwave energy delivery to tissues was quantified. Quantitative reverse transcriptase PCR was used to quantify mRNA expression. Immunohistochemistry and fluorescence immunostaining was used to assess protein expression. Microwave energy deposition induced sustained, localised cell death at the treatment site. There was a downregulation in levels of HPV oncoproteins E6 and E7 alongside a reduction in cellular growth/proliferation and induction of apoptosis/autophagy. HSP70 expression confirmed hyperthermia, concomitant with induction of translational stress. The data suggest that microwave treatment inhibits tumour cell proliferation and allows the natural apoptosis of HPV-infected cells to resume. Precision microwave delivery presents a potential new treatment for treating HPV-positive anogenital precancerous lesions and cancers. Funding was through an Innovate UK Biomedical Catalyst grant (ID# 92138-556187), a Chief Scientist Office grant (TCS/19/11) and core support from Medical Research Council (MC_ UU_12014) core funding for the MRC-University of Glasgow Centre for Virus Research.

HPV16 E6 induces chromosomal instability due to polar chromosomes caused by E6AP-dependent degradation of the mitotic kinesin CENP-E

Chromosome segregation during mitosis is highly regulated to ensure production of genetically identical progeny. Recurrent mitotic errors cause chromosomal instability (CIN), a hallmark of tumors. The E6 and E7 oncoproteins of high-risk human papillomavirus (HPV), which causes cervical, anal, and head and neck cancers (HNC), cause mitotic defects consistent with CIN in models of anogenital cancers, but this has not been studied in the context of HNC. Here, we show that HPV16 induces a specific type of CIN in patient HNC tumors, patient-derived xenografts, and cell lines, which is due to defects in chromosome congression. These defects are specifically induced by the HPV16 oncogene E6 rather than E7. We show that HPV16 E6 expression causes degradation of the mitotic kinesin CENP-E, whose depletion produces chromosomes that are chronically misaligned near spindle poles (polar chromosomes) and fail to congress. Though the canonical oncogenic role of E6 is the degradation of the tumor suppressor p53, CENP-E degradation and polar chromosomes occur independently of p53. Instead, E6 directs CENP-E degradation in a proteasome-dependent manner via the E6-associated ubiquitin protein ligase E6AP/UBE3A. This study reveals a mechanism by which HPV induces CIN, which may impact HPV-mediated tumor initiation, progression, and therapeutic response.