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/ß-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.

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

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

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.

E6-Encoded by Cancer-Causing Human Papillomavirus Interacts with Aurora Kinase A To Promote HPV-Mediated Carcinogenesis.

The expression of human papillomavirus (HPV) oncoproteins perturbed multiple cellular events of the host cells, leading to the formation of cancer phenotypes. Our current and previous studies indicated that Aurora kinase A (AurA), a mitotic regulator that is often aberrantly expressed in human cancers, is preferentially bound to E6-encoded by cancer-causing HPV. AurA is believed to be important for the proliferation and survival of HPV-positive cells. Nonetheless, the interaction between AurA and E6, and the mechanism of how this association is involved in carcinogenesis, have not been elucidated clearly. Hence, we performed a series of biochemical assays to characterize the AurA-E6 association and complex formation. We found the C-terminus of E6, upstream of the PDZ binding motif of E6, is important to forming the AurA-E6 complex in the nucleus. We also showed that the expression level of E6 corresponded positively with AurA expression. Meanwhile, the functional consequences of the AurA-E6 association to AurA kinase function and host cellular events were also delineated. Intriguingly, we revealed that AurA-E6 association regulated the expression of cyclin E and phosphor-Histone H3, which are involved in G1/S and mitotic phases of the cell cycle, respectively. Depletion of AurA also reduced the invasive ability of HPV-positive cells. AurA inhibition may not be sufficient to reduce the oncogenic potential exerted by E6. Altogether, our study unleashed the mechanism of how HPVE6 deploy AurA to promote cancer phenotypes, particularly through dysregulation of cell cycle checkpoints and suggests that the AurA-E6 complex possesses a therapeutic value. IMPORTANCE We unveiled the mechanism of how HPV employs Aurora kinase A (AurA) of host cells to exert its oncogenic capability synergistically. We systematically characterized the mode of interaction between E6-encoded by cancer-causing HPV and AurA. Then, we delineated the consequences of AurA-E6 complex formation on AurA kinase function and changes to cellular events at molecular levels. Using a cell-based approach, we unleashed that disruption of AurA-E6 association can halt cancer phenotype exhibited by HPV-positive cancer cells. Our findings are vital for the designing of state-of-the-art therapies for HPV-associated cancers.

Characterization of oral microbiota in HPV and non-HPV head and neck squamous cell carcinoma and its association with patient outcomes.

OBJECTIVE: To investigate the interplay among the oral microbiota, HPV infection, traditional risk factors and patient outcomes in head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: A multi-center study of HNSCC patients with paired tumor and control tissues. We characterized the oral microbiota and HPV infection of tissues in 166 Chinese adults by sequencing the bacterial 16S rRNA V3-V4 and HPV L1 regions, respectively, and examined the associations among the oral microbiota, HPV and clinical features. RESULTS: A total of 15.7% of the surveyed HNSCC patients were positive for HPV DNA, with infection rates varying from 66.7% in oropharyngeal SCC to 10.4% in oral cavity SCC (OSCC). No HPV infection was detected in the surveyed hypopharyngeal SCC. HPV16 was largely the predominant type. HPV infection in non-OSCC, especially oropharyngeal SCC, was associated with advanced N stage and superior survival outcomes. Oral microbiota dysbiosis was observed in HNSCC tumors, with differentially abundant taxa mainly associated with HNSCC subtype, T stage, survival/relapse, HPV infection, and smoking. Notably, the enrichment of Fusobacterium in tumor tissues of OSCC patients was associated with no smoking, early T stage, early N stage, and better 3-year disease-specific survival. CONCLUSION: Our findings underscore the involvement of oral microbiota dysbiosis in OSCC pathogenesis, Fusobacterium is involved with improved OSCC patient outcomes, especially in patients lacking traditional risk factors. Understanding the complex interactions among the oral microbiota, HPV infection and other risk factors for HNSCC will provide important insights into the pathogenesis of HNSCC.

Review of the Standard and Advanced Screening, Staging Systems and Treatment Modalities for Cervical Cancer.

Cancer arising from the uterine cervix is the fourth most common cause of cancer death among women worldwide. Almost 90% of cervical cancer mortality has occurred in low- and middle-income countries. One of the major aetiologies contributing to cervical cancer is the persistent infection by the cancer-causing types of the human papillomavirus. The disease is preventable if the premalignant lesion is detected early and managed effectively. In this review, we outlined the standard guidelines that have been introduced and implemented worldwide for decades, including the cytology, the HPV detection and genotyping, and the immunostaining of surrogate markers. In addition, the staging system used to classify the premalignancy and malignancy of the uterine cervix, as well as the safety and efficacy of the various treatment modalities in clinical trials for cervical cancers, are also discussed. In this millennial world, the advancements in computer-aided technology, including robotic modules and artificial intelligence (AI), are also incorporated into the screening, diagnostic, and treatment platforms. These innovations reduce the dependence on specialists and technologists, as well as the work burden and time incurred for sample processing. However, concerns over the practicality of these advancements remain, due to the high cost, lack of flexibility, and the judgment of a trained professional that is currently not replaceable by a machine.

Oesophageal carcinoma: The prevalence of DNA tumour viruses and therapy.

Oesophageal carcinoma ranks the sixth leading cause of cancer death and affected 544,000 - 604,000 people in 2020. Patients often presented with a poor cancer prognosis with a low survival rate of 15-25%. Depending upon the cell type, oesophageal carcinoma is categorised into oesophageal squamous cell carcinoma (ESCC) and oesophageal adenocarcinoma (EAC). ESCC is predominantly reported in developing countries, while EAC is more common in developed countries. Aside from the presence of exogenous co-factors, such as cigarette smoking, alcohol consumption, obesity, gastroesophageal reflux disease (GERD); infection with oncogenic viruses is suspected to be one of the major factors contributing to EC development. Oncogenic viruses, including human papillomavirus (HPV), Epstein Barr virus (EBV), Cytomegalovirus (CMV) and Herpes Simplex Virus (HSV) have been detected in various proportions of EC samples. Nonetheless, their aetiological roles in EC remain debatable. In this review, we garnered previous studies that focus on the association between oncogenic viruses and EC. Among these oncogenic viruses, HPV appears to have a stronger association with EC than the others. In addition, we also discuss the pros and cons of the treatment regimens to treat EC patients, including immunotherapy, chemo- and chemoradiotherapy, and their efficacy.

Current Updates on Cancer-Causing Types of Human Papillomaviruses (HPVs) in East, Southeast, and South Asia.

Human papillomavirus (HPV) infection remains one of the most prominent cancer-causing DNA viruses, contributing to approximately 5% of human cancers. While association between HPV and cervical cancers has been well-established, evidence on the attribution of head and neck cancers (HNC) to HPV have been increasing in recent years. Among the cancer-causing HPV genotypes, HPV16 and 18 remain the major contributors to cancers across the globe. Nonetheless, the distribution of HPV genotypes in ethnically, geographically, and socio-economically diverse East, Southeast, and South Asia may differ from other parts of the world. In this review, we garner and provide updated insight into various aspects of HPV reported in recent years (2015-2021) in these regions. We included: (i) the HPV genotypes detected in normal cancers of the uterine cervix and head and neck, as well as the distribution of the HPV genotypes by geography and age groups; (ii) the laboratory diagnostic methods and treatment regimens used within these regions; and (iii) the oncogenic properties of HPV prototypes and their variants contributing to carcinogenesis. More importantly, we also unveil the similarities and discrepancies between these aspects, the areas lacking study, and the challenges faced in HPV studies.

The Intersection between Oral Microbiota, Host Gene Methylation and Patient Outcomes in Head and Neck Squamous Cell Carcinoma.

The role of oral microbiota in head and neck squamous cell carcinoma (HNSCC) is poorly understood. Here we sought to evaluate the association of the bacterial microbiome with host gene methylation and patient outcomes, and to explore its potential as a biomarker for early detection or intervention. Here we performed 16S rRNA gene amplicon sequencing in sixty-eight HNSCC patients across both tissue and oral rinse samples to identify oral bacteria with differential abundance between HNSCC and controls. A subset of thirty-one pairs of HNSCC tumor tissues and the adjacent normal tissues were characterized for host gene methylation profile using bisulfite capture sequencing. We observed significant enrichments of Fusobacterium and Peptostreptococcus in HNSCC tumor tissues when compared to the adjacent normal tissues, and in HNSCC oral rinses when compared to healthy subjects, while ten other bacterial genera were largely depleted. These HNSCC-related bacteria were discriminative for HNSCC and controls with area under the receiver operating curves (AUCs) of 0.84 and 0.86 in tissue and oral rinse samples, respectively. Moreover, Fusobacterium nucleatum abundance in HNSCC cases was strongly associated with non-smokers, lower tumor stage, lower rate of recurrence, and improved disease-specific survival. An integrative analysis identified that enrichment of F. nucleatum was associated with host gene promoter methylation, including hypermethylation of tumor suppressor genes LXN and SMARCA2, for which gene expressions were downregulated in the HNSCC cohort from The Cancer Genome Atlas. In conclusion, we identified a taxonomically defined microbial consortium associated with HNSCC that may have clinical potential regarding biomarkers for early detection or intervention. Host-microbe interactions between F. nucleatum enrichment and clinical outcomes or host gene methylation imply a potential role of F. nucleatum as a pro-inflammatory driver in initiating HNSCC without traditional risk factors, which warrants further investigation for the underlying mechanisms.

Human Papillomavirus 58 E7 T20I/G63S Variant Isolated from an East Asian Population Possesses High Oncogenicity.

Human papillomavirus (HPV) type 58 is the third most commonly detected HPV type in cervical cancer among Eastern Asians. Our previous international epidemiological studies revealed that HPV58 carrying an E7 natural variant, T20I/G63S (designated V1), was associated with a higher risk of cervical cancer. We recently showed that V1 possesses a greater ability to immortalize and transform primary cells, as well as degrading pRB more effectively, than the prototype and other common variants. In this study, we performed a series of phenotypic and molecular assays using physiologically relevant in vitro and in vivo models to compare the oncogenicity of V1 with that of the prototype and other common natural variants. Through activation of the AKT and K-Ras/extracellular signal-regulated kinase (ERK) signaling pathways, V1 consistently showed greater oncogenicity than the prototype and other variants, as demonstrated by increased cell proliferation, migration, and invasion, as well as induction of larger tumors in athymic nude mice. This study complements our previous epidemiological and molecular observations pinpointing the higher oncogenicity of V1 than that of the prototype and all other common variants. Since V1 is more commonly found in eastern Asia, our report provides insight into the design of HPV screening assays and selection of components for HPV vaccines in this region.IMPORTANCE Epidemiological studies have revealed that a wild-type variant of HPV58 carrying an E7 variation, T20I/G63S (V1), is associated with a higher risk of cervical cancer. We previously reported that this increased oncogenicity could be the result of the virus's greater ability to degrade pRB, thereby leading to an increased ability to grow in an anchorage-independent manner. In addition to this, this report further showed that this HPV variant induced activation of the AKT and K-Ras/ERK signaling pathways, thereby explaining its genuine oncogenicity in promoting cell proliferation, migration, invasion, and formation of tumors, all to a greater extent than the prototype HPV58 and other common variants.

Human papillomavirus type 18 oncoproteins exert their oncogenicity in esophageal and tongue squamous cell carcinoma cell lines distinctly.

BACKGROUND: Increasing evidence indicates an etiological role of human papillomavirus (HPV) in head and neck cancers, particularly oropharyngeal squamous cell carcinoma (OPSCC). However, the association between HPV and other cancers, including esophageal and tongue remains unclear. This study delineated the molecular characteristics of HPV18 E6 and E7 in esophageal (EC109 and EC9706) and tongue (Tca83) cancer cell lines with reference to cervical cancer (HeLa). METHODS: We analysed the HPV transcription profiles of esophageal and tongue cancer cells through Next-generation RNA sequencing, and the role of HPV18 E6 and E7 in these cells was assessed via siRNA approach, Western blotting and immunofluorescence assays. RESULTS: Overall, the HPV transcription profiles of esophageal and tongue cancer cells mimicked that of cervical cancer cells, with notable disruption of E2, and expression of E6, spliced E6 (E6*), E7, E1 and L1 transcripts. As with cervical cancer cells, p53 and its downstream transactivation target, p21, were found to be the major targets of E6 in esophageal and tongue cancer cell lines. Intriguingly, E7 preferentially targeted p130 in the two esophageal cancer cell lines, instead of pRb as in cervical cancer. Tca83 exhibited an E7 to E6 transcript ratio comparable to HeLa (cervix), targeted the ERK1/2 and MMP2 pathways, and was dependent on E6 and E7 to survive and proliferate. In contrast, both the esophageal cancer cell lines were distinct from HeLa in these aspects. CONCLUSIONS: This is the first study that delineates transcript expression and protein interaction of HPV18 E6 and E7 in esophageal and tongue cancer cell lines, suggesting that HPV plays a role in inducing these cancers, albeit via distinct pathways than those observed in cervical cancer.

Correction to: Human papillomavirus type 18 oncoproteins exert their oncogenicity in esophageal and tongue squamous cell carcinoma cell lines distinctly.

Following publication of the original article [1], the authors reported that during the production process, Table 1 was omitted.

Role of polycyclic aromatic hydrocarbons as a co-factor in human papillomavirus-mediated carcinogenesis.

BACKGROUND: Human papillomavirus (HPV) is an etiological agent of cervical cancer. Yet co-factors are believed to be involved in HPV-mediated carcinogenesis. Polycyclic aromatic hydrocarbons (PAHs) are considered as one of these co-factors. Epidemiologic studies have associated high PAH exposure with increased risk for cancer development. To date, many studies focus on benzo[a]pyrene, however, the role of other PAHs should not be neglected. This study aimed to compare the potential of different PAHs as a co-factor in HPV-mediated carcinogenesis, and to investigate the possible mechanisms involved. METHODS: The effect of 17 PAHs on high-risk HPV (HPV16) were examined in this study. HPV16 E7 oncogene was expressed in primary cells extracted from baby rat kidney and treated with PAHs. The co-transforming ability of PAHs were measured by colony formation index according to the number and size of transformed colonies. Effects of PAHs on proliferation of HPV-null (C33A) and -infected (CaSki) were examined using CCK-8 assay. Wound healing assay and matrigel invasion chambers were used to investigate effects of PAHs on cell motility and invasivion of HPV-null (MCF7, C33A) and -infected (SiHa) cells. RESULTS: Benzo[a]pyrene (BaP), dibenz[a,h]anthracene (DBA) and indeno[1,2,3-cd]pyrene (IDP) showed the greatest co-transforming potential in the baby rat kidney cell system. Short-term exposure to BaP, DBA, IDP and pyrene (PR) did not affect proliferation of C33A or CaSki cells, however, long-term exposure of these four PAHs led to dramatic increase in growth rate of CaSki cells by 120-140%. Besides, exposure of PAHs has an effect on cell motility and invasiveness of C33A and SiHa cells, but not for MCF7 cells. Exposure of BaP and DBA enhanced migration (1.26 to 1.40-fold) and invasion (1.68 to 1.94-fold) capacity of C33A cells. Intriguingly, exposure of all four types of PAHs boosted the migration (1.12 to 1.28-fold) and invasion (1.26 to 1.40-fold) capacity of SiHa cells. CONCLUSIONS: Our results indicate that exposure to PAHs can be a key co-factor in HPV-related cancer development. They could act on all three stages, namely initiation, promotion and progression. Further study is needed to unveil the mechanisms by which PAHs interact with HPV to cause malignancy.

Oncogenicitiy Comparison of Human Papillomavirus Type 52 E6 Variants.

Human papillomavirus (HPV) infection contributes to virtually all cases of cervical cancer, the fourth most common cancer affecting women worldwide. The oncogenicity of HPV is mainly attributable to the E6 and E7 oncoproteins. HPV-52 is the seventh most common HPV type globally, but it has a remarkably high prevalence in East Asia. In previous studies it has been speculated that the oncogenicity might vary among different HPV-52 variants. In the present study, we compared the oncogenicity of E6 derived from the HPV-52 prototype and three commonly found variants, V1 (K93R), V2 (E14D/V92L) and V3 (K93R/N122K), through molecular and phenotypic approaches. We demonstrated that cells containing V1 achieved higher colony formation and showed greater cell migration ability when compared to other variants, but no difference in cell immortalization ability was observed. At the molecular level, the three variants formed complexes with E6-associated protein (E6AP) and p53 as efficiently as the prototype. They degraded p53 and PSD95/Dlg/ZO-1(PDZ) proteins, including MAGI-1c and Dlg, to a similar extent. They also exhibited a similar subcellular localization, and shared a half-life of approximately 45 min. Our findings provide a clearer picture of HPV-52 E6 variant oncogenicity, which is important for further studies aiming to understand the unusually high prevalence of HPV-52 among cervical cancers in East Asia.

Oncogenic comparison of human papillomavirus type 58 E7 variants.

Human papillomavirus 58 (HPV58) ranks the second or third in East Asian cervical cancers. Current studies on HPV58 are scarce and focus on the prototype. Previously, we identified the three most common circulating HPV58 E7 strains contained amino acid alterations: G41R/G63D (51%), T20I/G63S (22%) and T74A/D76E (14%) respectively. Among them, the T20I/G63S variant (V1) had a stronger epidemiological association with cervical cancer. We therefore suggested that V1 possessed stronger oncogenicity than the other two variants. Here, we performed phenotypic assays to characterize and compare their oncogenicities with HPV58 E7 prototype. Our results showed that overexpression of V1 conferred a higher colony-forming ability to primary murine epithelial cells than prototype (P < 0.05) and other variants, implicating its higher immortalising potential. Further experiments showed that both V1 and prototype enhanced the anchorage-independent growth of NIH/3T3 cells (P < 0.001), implicating their stronger transforming power than the two other variants. Moreover, they possessed an increased ability to degrade pRb (P < 0.001), which is a major effector pathway of E7-driven oncogenesis. Our work represents the first study to compare the oncogenicities of HPV58 E7 prototype and variants. These findings deepened our understanding of HPV58 and might inform clinical screening and follow-up strategy.

The Human Papillomavirus E6 PDZ Binding Motif Links DNA Damage Response Signaling to E6 Inhibition of p53 Transcriptional Activity.

The presence of a PDZ binding motif (PBM) in the human papillomavirus (HPV) E6 oncoprotein appears to be a characteristic marker of high oncogenic potential and confers interaction with a number of different cellular PDZ domain-containing substrates. The E6 PBM is also subject to phosphorylation, resulting in inhibition of E6 PDZ binding activity and instead allowing E6 to associate with 14-3-3 proteins. In this study, we analyzed the conditions under which the E6 PBM is phosphorylated. We demonstrate that in normal cycling cells, the levels of E6 phosphorylation are very low. However, following exposure of cells to oxidative stress or the induction of DNA damage, there is a striking increase in the levels of E6 phosphorylation. Depending on the specific stimulus, this phosphorylation of E6 can involve the ATM/ATR pathway and is performed primarily through Chk1, although the Chk2 pathway is also involved indirectly through activation of protein kinase A (PKA). To understand the biological relevance of these phospho-modifications of E6, we analyzed their effects upon the ability of E6 to inhibit p53 transcriptional activity. We show that an intact E6 phospho-acceptor site plays an essential role in the ability of E6 to inhibit p53 transcriptional activity on a subset of p53-responsive promoters in a manner that is independent of E6's ability to direct p53 degradation. These results are, to our knowledge, the first example of a DNA damage response controlling PBM-PDZ recognition. This study also provides links between the DNA damage response, the regulation of E6 PBM function, and the inhibition of p53 activity and begins to explain how HPV-infected cells remain within the cell cycle, despite activation of DNA damage response pathways during productive virus infections.IMPORTANCE The cancer-causing HPV E6 oncoproteins all possess a PDZ binding motif at their extreme carboxy termini. Depending upon whether this motif is phosphorylated, E6 can recognize PDZ domain-containing proteins or members of the 14-3-3 family of proteins. We show here that DNA damage response pathways directly signal to the E6 PBM, resulting in Chk1- and Chk2-driven phosphorylation. This phosphorylation is particularly pronounced following treatment of cells with a variety of different chemotherapeutic drugs. A direct functional consequence of this signaling is to confer an enhanced ability upon E6 to inhibit p53 transcriptional activity in a proteasome-independent but phosphorylation-dependent manner. These results are the first example of DNA damage signaling pathways regulating PBM-PDZ interactions and provide the mechanistic link between E6 PBM function and perturbation of p53 activity.

Ancient Evolution and Dispersion of Human Papillomavirus 58 Variants.

Human papillomavirus 58 (HPV58) is found in 10 to 18% of cervical cancers in East Asia but is rather uncommon elsewhere. The distribution and oncogenic potential of HPV58 variants appear to be heterogeneous, since the E7 T20I/G63S variant is more prevalent in East Asia and confers a 7- to 9-fold-higher risk of cervical precancer and cancer. However, the underlying genomic mechanisms that explain the geographic and carcinogenic diversity of HPV58 variants are still poorly understood. In this study, we used a combination of phylogenetic analyses and bioinformatics to investigate the deep evolutionary history of HPV58 complete genome variants. The initial splitting of HPV58 variants was estimated to occur 478,600 years ago (95% highest posterior density [HPD], 391,000 to 569,600 years ago). This divergence time is well within the era of speciation between Homo sapiens and Neanderthals/Denisovans and around three times longer than the modern Homo sapiens divergence times. The expansion of present-day variants in Eurasia could be the consequence of viral transmission from Neanderthals/Denisovans to non-African modern human populations through gene flow. A whole-genome sequence signature analysis identified 3 amino acid changes, 16 synonymous nucleotide changes, and a 12-bp insertion strongly associated with the E7 T20I/G63S variant that represents the A3 sublineage and carries higher carcinogenetic potential. Compared with the capsid proteins, the oncogenes E7 and E6 had increased substitution rates indicative of higher selection pressure. These data provide a comprehensive evolutionary history and genomic basis of HPV58 variants to assist further investigation of carcinogenic association and the development of diagnostic and therapeutic strategies.IMPORTANCE Papillomaviruses (PVs) are an ancient and heterogeneous group of double-stranded DNA viruses that preferentially infect the cutaneous and mucocutaneous epithelia of vertebrates. Persistent infection by specific oncogenic human papillomaviruses (HPVs), including HPV58, has been established as the primary cause of cervical cancer. In this work, we reveal the complex evolutionary history of HPV58 variants that explains the heterogeneity of oncogenic potential and geographic distribution. Our data suggest that HPV58 variants may have coevolved with archaic hominins and dispersed across the planet through host interbreeding and gene flow. Certain genes and codons of HPV58 variants representing higher carcinogenic potential and/or that are under positive selection may have important implications for viral host specificity, pathogenesis, and disease prevention.

Increased Growth of a Newly Established Mouse Epithelial Cell Line Transformed with HPV-16 E7 in Diabetic Mice.

Epidemiological evidence supports that infection with high-risk types of human papillomavirus (HPV) can interact with host and environmental risk factors to contribute to the development of cervical, oropharyngeal, and other anogenital cancers. In this study, we established a mouse epithelial cancer cell line, designated as Chinese University Papillomavirus-1 (CUP-1), from C57BL/KsJ mice through persistent expression of HPV-16 E7 oncogene. After continuous culturing of up to 200 days with over 60 passages, we showed that CUP-1 became an immortalized and transformed epithelial cell line with continuous E7 expression and persistent reduction of retinoblastoma protein (a known target of E7). This model allowed in-vivo study of interaction between HPV and co-factors of tumorigenesis in syngeneic mice. Diabetes has been shown to increase HPV pathogenicity in different pathological context. Herein, with this newly-established cell line, we uncovered that diabetes promoted CUP-1 xenograft growth in syngeneic db/db mice. In sum, we successfully established a HPV-16 E7 transformed mouse epithelial cell line, which allowed subsequent studies of co-factors in multistep HPV carcinogenesis in an immunocompetent host. More importantly, this study is the very first to demonstrate the promoting effect of diabetes on HPV-associated carcinogenesis in vivo, implicating the importance of cancer surveillance in diabetic environment.

Inhibitor of caspase-activated DNase expression enhances caspase-activated DNase expression and inhibits oxidative stress-induced chromosome breaks at the mixed lineage leukaemia gene in nasopharyngeal carcinoma cells.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is commonly found in Asia, especially among the Chinese ethnic group. Chromosome rearrangements are common among NPC patients. Although the mechanism underlying the chromosome rearrangements in NPC is unclear, various mechanisms including activation of caspase-activated DNase (CAD) were proposed to contribute to chromosome rearrangements in leukaemia. Activation of CAD can be initiated by multiple agents, including oxidative stress, which is well implicated in carcinogenesis. CAD is the main enzyme that causes DNA fragmentation during apoptosis, and CAD is also implicated in promoting cell differentiation. In view of the role of oxidative stress in carcinogenesis and CAD activation, and since CAD was suggested to contribute to chromosome rearrangement in leukaemia, we hypothesise that oxidative stress-induced CAD activation could be one of the mechanisms that leads to chromosome rearrangements in NPC. METHODS: SUNEI cells were treated with various concentrations of H2O2 for different period of time to ensure that cells undergo H2O2-induced MLL gene cleavage. Transfections with hCAD, mCAD, mutant hCAD, or cotransfection with hCAD and mICAD, and cotransfection with mutant hCAD and mICAD were performed. Gene expression was confirmed by Western blotting and MLL gene cleavage was assessed by inverse polymerase chain reaction (IPCR). RESULTS: Treatment with H2O2 clearly induces cleavages within the MLL gene which locates at 11q23, a common deletion site in NPC. In order to investigate the role of CAD, CAD was overexpressed in SUNE1 cells, but that did not result in significant changes in H2O2-induced MLL gene cleavage. This could be because CAD requires ICAD for proper folding. Indeed, by overexpressing ICAD alone or co-expressing ICAD with CAD, Western blotting showed that CAD was expressed. In addition, ICAD overexpression also suppressed H2O2-induced MLL gene cleavage, suggesting a possible role of CAD in initiating chromosome cleavage during oxidative stress. CONCLUSIONS: Oxidative stress mediated by H2O2 induces cleavage of the MLL gene, most likely via the caspase-activated DNase, CAD, and CAD expression requires ICAD. Since the MLL gene is located at 11q23, a common deletion site in NPC, thus stress-induced CAD activation may represent one of the mechanisms leading to chromosome rearrangement in NPC.

Cancer-causing human papillomavirus E6 proteins display major differences in the phospho-regulation of their PDZ interactions.

UNLABELLED: Previous studies have shown that the cancer-causing high-risk human papillomavirus (HPV) E6 oncoproteins have PDZ binding potential, an activity which is important for their ability to support the viral life cycle and to cooperate in the induction of malignancy. However, PDZ interactions are not constitutive, and they can be negatively regulated by phosphorylation within the E6 PDZ binding motif (PBM). In this study, we have investigated the differential regulation of the HPV E6 PBMs from diverse high-risk HPV types. We show that, depending on the HPV type, PDZ binding activity can be regulated by phosphorylation with protein kinase A (PKA) or AKT, which, in turn, inhibits PDZ recognition. Such regulation is highly conserved between E6 proteins derived from HPV-16, HPV-18, and HPV-58 while being somewhat weaker or absent from other types such as HPV-31, HPV-33, and HPV-51. In the case of HPV31, PKA phosphorylation occurs within the core of the E6 protein and has no effect on PDZ interactions, and this demonstrates a surprising degree of heterogeneity among the different high-risk HPV E6 oncoproteins in how they are regulated by different cellular signaling pathways. IMPORTANCE: This study demonstrated that the cancer-causing HPV E6 oncoproteins are all subject to posttranslational modification of their extreme C-terminal PDZ binding motifs through phosphorylation. However, the identities of the kinase are not the same for all HPV types. This demonstrates a very important divergence between these HPVs, and it suggests that changes in cell signaling pathways have different consequences for different high-risk virus infections and their associated malignancies.