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

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

Three-dimensional chromatin analysis reveals Sp1 as a mediator to program and reprogram HPV-host epigenetic architecture in cervical cancer.

Human papillomavirus (HPV) is predominantly associated with HPV-related cancers, however, the precise mechanisms underlying the HPV-host epigenetic architectures in HPV carcinogenesis remain elusive. Here, we employed high-throughput chromosome conformation capture (Hi-C) to comprehensively map HPV16/18-host chromatin interactions. Our study identified the transcription factor Sp1 as a pivotal mediator in programming HPV-host interactions. By targeting Sp1, the active histone modifications (H3K27ac, H3K4me1, and H3K4me3) and the HPV-host chromatin interactions are reprogrammed, which leads to the downregulation of oncogenes located near the integration sites in both HPV (E6/E7) and the host genome (KLF5/MYC). Additionally, Sp1 inhibition led to the upregulation of immune checkpoint genes by reprogramming histone modifications in host cells. Notably, humanized patient-derived xenograft (PDX-HuHSC-NSG) models demonstrated that Sp1 inhibition promoted anti-PD-1 immunotherapy via remodeling the tumor immune microenvironment in cervical cancer. Moreover, single-cell transcriptomic analysis validated the enrichment of transcription factor Sp1 in epithelial cells of cervical cancer. In summary, our findings elucidate Sp1 as a key mediator involved in the programming and reprogramming of HPV-host epigenetic architecture. Inhibiting Sp1 with plicamycin may represent a promising therapeutic option for HPV-related carcinoma.

MAL expression downregulation through suppressive H3K27me3 marks at the promoter in HPV16-related cervical cancers is prognostically relevant and manifested by the interplay of novel MAL antisense long noncoding RNA AC103563.8, E7 oncoprotein and EZH2.

BACKGROUND: MAL (T-lymphocyte maturation-associated protein) is highly downregulated in most cancers, including cervical cancer (CaCx), attributable to promoter hypermethylation. Long noncoding RNA genes (lncGs) play pivotal roles in CaCx pathogenesis, by interacting with human papillomavirus (HPV)-encoded oncoproteins, and epigenetically regulating coding gene expression. Hence, we attempted to decipher the impact and underlying mechanisms of MAL downregulation in HPV16-related CaCx pathogenesis, by interrogating the interactive roles of MAL antisense lncRNA AC103563.8, E7 oncoprotein and PRC2 complex protein, EZH2. RESULTS: Employing strand-specific RNA-sequencing, we confirmed the downregulated expression of MAL in association with poor overall survival of CaCx patients bearing HPV16, along with its antisense long noncoding RNA (lncRNA) AC103563.8. The strength of positive correlation between MAL and AC103563.8 was significantly high among patients compared to normal individuals. While downregulated expression of MAL was significantly associated with poor overall survival of CaCx patients bearing HPV16, AC103563.8 did not reveal any such association. We confirmed the enrichment of chromatin suppressive mark, H3K27me3 at MAL promoter, using ChIP-qPCR in HPV16-positive SiHa cells. Subsequent E7 knockdown in such cells significantly increased MAL expression, concomitant with decreased EZH2 expression and H3K27me3 marks at MAL promoter. In silico analysis revealed that both E7 and EZH2 bear the potential of interacting with AC103563.8, at the same binding domain. RNA immunoprecipitation with anti-EZH2 and anti-E7 antibodies, respectively, and subsequent quantitative PCR analysis in E7-silenced and unperturbed SiHa cells confirmed the interaction of AC103563.8 with EZH2 and E7, respectively. Apparently, AC103563.8 seems to preclude EZH2 and bind with E7, failing to block EZH2 function in patients. Thereby, enhanced EZH2 expression in the presence of E7 could potentially inactivate the MAL promoter through H3K27me3 marks, corroborating our previous results of MAL expression downregulation in patients. CONCLUSION: AC103563.8-E7-EZH2 axis, therefore, appears to crucially regulate the expression of MAL, through chromatin inactivation in HPV16-CaCx pathogenesis, warranting therapeutic strategy development.

Human papillomavirus-16 E6 activates the pentose phosphate pathway to promote cervical cancer cell proliferation by inhibiting G6PD lactylation.

High-risk human papillomaviruses (HPVs) are the causative agents of cervical cancer. Here, we report that HPV16 E6E7 promotes cervical cancer cell proliferation by activating the pentose phosphate pathway (PPP). We found that HPV16 E6 activates the PPP primarily by increasing glucose-6-phosphate dehydrogenase (G6PD) enzyme activity. Mechanistically, HPV16 E6 promoted G6PD dimer formation by inhibiting its lactylation. Importantly, we suggest that G6PD K45 was lactylated during G6PD-mediated antioxidant stress. In primary human keratinocytes and an HPV-negative cervical cancer C33A cells line ectopically expressing HPV16 E6, the transduction of G6PD K45A (unable to be lactylated) increased GSH and NADPH levels and, correspondingly, decreasing ROS levels. Conversely, the re-expression of G6PD K45T (mimicking constitutive lactylation) in HPV16-positive SiHa cells line inhibited cell proliferation. In vivo, the inhibition of G6PD enzyme activity with 6-aminonicotinamide (6-An) or the re-expression of G6PD K45T inhibited tumor proliferation. In conclusion, we have revealed a novel mechanism of HPV oncoprotein-mediated malignant transformation. These findings might provide effective strategies for treating cervical and HPV-associated cancers.

Alterations of UHRF family Expression and was regulated by High Risk Type HPV16 in Uterine Cervical Cancer.

The altered protein expression of inverted CCAAT box-binding protein of 90 kDa/ubiquitin-like with PHD and RING finger domains 1 (ICBP90/UHRF1), and Np95-like ring finger protein (NIRF)/UHRF2, which belong to the ubiquitin-like with PHD and RING finger domains (UHRF) family, is linked to tumor malignancy and the progression of various cancers. In this study, we analyzed the UHRF family expression in cervical cancers, and it's regulation by human papillomavirus (HPV). Western blotting was performed to analyze protein expression in cervical cancer cell lines. Immunohistochemical analysis were used to investigate the expression of UHRF family and MIB-1 in cervical cancer tissues. Transfection were done for analyze the relationship between UHRF family and HPVs. We showed that NIRF expression was decreased and ICBP90 expression was increased in cervical cancers compared to normal counterparts. Western blotting also showed that NIRF expression was quite low levels, but ICBP90 was high in human cervical cancer cell lines. Interestingly, ICBP90 was up regulated by high risk type HPV16 E6 and E7, but not low-risk type HPV11. On the other hand, NIRF was down regulated by high risk type HPV16 E6 but not by E7. Low risk type HPV11 E6 did not affect the NIRF expression at all. We propose that ICBP90 overexpression, and reduced NIRF expression, found in cervical cancers, is an important event of a cervical carcinogenesis, and especially ICBP90 may offer a proliferating marker and therapeutic target for treating uterine cervical cancers.

ErbB2/HER2 receptor tyrosine kinase regulates human papillomavirus promoter activity.

Human papillomaviruses (HPVs) are a major cause of cancer. While surgical intervention remains effective for a majority of HPV-caused cancers, the urgent need for medical treatments targeting HPV-infected cells persists. The pivotal early genes E6 and E7, which are under the control of the viral genome's long control region (LCR), play a crucial role in infection and HPV-induced oncogenesis, as well as immune evasion. In this study, proteomic analysis of endosomes uncovered the co-internalization of ErbB2 receptor tyrosine kinase, also called HER2/neu, with HPV16 particles from the plasma membrane. Although ErbB2 overexpression has been associated with cervical cancer, its influence on HPV infection stages was previously unknown. Therefore, we investigated the role of ErbB2 in HPV infection, focusing on HPV16. Through siRNA-mediated knockdown and pharmacological inhibition studies, we found that HPV16 entry is independent of ErbB2. Instead, our signal transduction and promoter assays unveiled a concentration- and activation-dependent regulatory role of ErbB2 on the HPV16 LCR by supporting viral promoter activity. We also found that ErbB2's nuclear localization signal was not essential for LCR activity, but rather the cellular ErbB2 protein level and activation status that were inhibited by tucatinib and CP-724714. These ErbB2-specific tyrosine kinase inhibitors as well as ErbB2 depletion significantly influenced the downstream Akt and ERK signaling pathways and LCR activity. Experiments encompassing low-risk HPV11 and high-risk HPV18 LCRs uncovered, beyond HPV16, the importance of ErbB2 in the general regulation of the HPV early promoter. Expanding our investigation to directly assess the impact of ErbB2 on viral gene expression, quantitative analysis of E6 and E7 transcript levels in HPV16 and HPV18 transformed cell lines unveiled a noteworthy decrease in oncogene expression following ErbB2 depletion, concomitant with the downregulation of Akt and ERK signaling pathways. In light of these findings, we propose that ErbB2 holds promise as potential target for treating HPV infections and HPV-associated malignancies by silencing viral gene expression.

Evaluation of the anti-cancer efficacy of lipid nanoparticles containing siRNA against HPV16 E6/E7 combined with cisplatin in a xenograft model of cervical cancer.

OBJECTIVE: To investigate the anti-cancer efficacy of ENB101-LNP, an ionizable lipid nanoparticles (LNPs) encapsulating siRNA against E6/E7 of HPV 16, in combination therapy with cisplatin in cervical cancer in vitro and in vivo. METHODS: CaSki cells were treated with ENB101-LNP, cisplatin, or combination. Cell viability assessed the cytotoxicity of the treatment. HPV16 E6/E7 gene knockdown was verified with RT-PCR both in vitro and in vivo. HLA class I and PD-L1 were checked by flow cytometry. A xenograft model was made using CaSki cells in BALB/c nude mice. To evaluate anticancer efficacy, mice were grouped. ENB101-LNP was given three times weekly for 3 weeks intravenously, and cisplatin was given once weekly intraperitoneally. Tumor growth was monitored. On day 25, mice were euthanized; tumors were collected, weighed, and imaged. Tumor samples were analyzed through histopathology, immunostaining, and western blot. RESULTS: ENB101-LNP and cisplatin synergistically inhibit CaSki cell growth. The combination reduces HPV 16 E6/E7 mRNA and boosts p21 mRNA, p53, p21, and HLA class I proteins. In mice, the treatment significantly blocked tumor growth and promoted apoptosis. Tumor inhibition rates were 29.7% (1 mpk ENB101-LNP), 29.6% (3 mpk), 34.0% (cisplatin), 47.0% (1 mpk ENB101-LNP-cisplatin), and 68.8% (3 mpk ENB101-LNP-cisplatin). RT-PCR confirmed up to 80% knockdown of HPV16 E6/E7 in the ENB101-LNP groups. Immunohistochemistry revealed increased p53, p21, and HLA-A expression with ENB101-LNP treatments, alone or combined. CONCLUSION: The combination of ENB101-LNP, which inhibits E6/E7 of HPV 16, with cisplatin, demonstrated significant anticancer activity in the xenograft mouse model of cervical cancer.

HIV-1 Reverse Transcriptase Expression in HPV16-Infected Epidermoid Carcinoma Cells Alters E6 Expression and Cellular Metabolism, and Induces a Hybrid Epithelial/Mesenchymal Cell Phenotype.

The high incidence of epithelial malignancies in HIV-1 infected individuals is associated with co-infection with oncogenic viruses, such as high-risk human papillomaviruses (HR HPVs), mostly HPV16. The molecular mechanisms underlying the HIV-1-associated increase in epithelial malignancies are not fully understood. A collaboration between HIV-1 and HR HPVs in the malignant transformation of epithelial cells has long been anticipated. Here, we delineated the effects of HIV-1 reverse transcriptase on the in vitro and in vivo properties of HPV16-infected cervical cancer cells. A human cervical carcinoma cell line infected with HPV16 (Ca Ski) was made to express HIV-1 reverse transcriptase (RT) by lentiviral transduction. The levels of the mRNA of the E6 isoforms and of the factors characteristic to the epithelial/mesenchymal transition were assessed by real-time RT-PCR. The parameters of glycolysis and mitochondrial respiration were determined using Seahorse technology. RT expressing Ca Ski subclones were assessed for the capacity to form tumors in nude mice. RT expression increased the expression of the E6*I isoform, modulated the expression of E-CADHERIN and VIMENTIN, indicating the presence of a hybrid epithelial/mesenchymal phenotype, enhanced glycolysis, and inhibited mitochondrial respiration. In addition, the expression of RT induced phenotypic alterations impacting cell motility, clonogenic activity, and the capacity of Ca Ski cells to form tumors in nude mice. These findings suggest that HIV-RT, a multifunctional protein, affects HPV16-induced oncogenesis, which is achieved through modulation of the expression of the E6 oncoprotein. These results highlight a complex interplay between HIV antigens and HPV oncoproteins potentiating the malignant transformation of epithelial cells.

The difference of transcriptome of HPV-infected patients contributes more to the occurrence of cervical cancer than the mutations of E6 and E7 genes in HPV16.

Human papillomavirus (HPV) E6 and E7 genes are biomarkers and drivers of the progression of cervical cancer (CxCa). The aim of this study was to investigate the relationship between HPV16 E6, E7 gene mutations and the occurrence and development of CxCa. Cervical exfoliated cells and clinical data of patients with cervical diseases were collected. Sample DNA was extracted, the E6 and E7 gene fragments were amplified by PCR, and the mutations were detected by Sanger sequencing and compared with standard sequences. Microarray was used to sequence the transcriptome of cells. Data of transcriptome analyzed and visualized using R software and its packages. Analysis of clinical characteristics demonstrated the association of HPV16 infection with CxCa (P < .05). Sanger sequencing results showed that the mutation sites of E6 gene included T178G/A, T350G, A131C, and T241G; among these, A131C and T241G were synonymous mutations. The mutation sites of E7 gene included A647G, T846C, G666A, T843C, and T760C, and all of them were synonymous mutations except A647G. There was no significant difference in the distribution of HPV16 E6, E7 mutations among CxCa, cervical intraepithelial neoplasia, and infection groups (P > .05). Compared with the non- CxCa group, gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes (DEGs) showed more significant enrichment of DEGs in the biological processes, pathways, and diseases closely related to cancer. Compared with the non-mutation group, the DEGs in the E6, E7 gene mutation group were significantly enriched in the events related to infection and immunity. To summarize, HPV16 may be associated with the occurrence and development of CxCa, but HPV16 E6 and E7 gene mutations have little effect on the occurrence and development of CxCa. Individual differences may have a greater effect on the progression of CxCa.

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.

A novel HPV16 splicing enhancer critical for viral oncogene expression and cell immortalization.

High-risk carcinogenic human papillomaviruses (HPVs), e.g. HPV16, express the E6 and E7 oncogenes from two mRNAs that are generated in a mutually exclusive manner by splicing. The HPV16 E7 mRNA, also known as the E6*I/E7 mRNA, is produced by splicing between splice sites SD226 and SA409, while E6 mRNAs retain the intron between these splice sites. We show that splicing between HPV16 splice sites SD226 and SA409 is controlled by a splicing enhancer consisting of a perfect repeat of an adenosine-rich, 11 nucleotide sequence: AAAAGCAAAGA. Two nucleotide substitutions in both 11 nucleotide sequences specifically inhibited production of the spliced E6*I/E7 mRNA. As a result, production of E7 protein was reduced and the ability of HPV16 to immortalize human primary keratinocytes was abolished. The splicing-enhancing effect was mediated by the cellular TRAP150/THRAP3 protein that also enhanced splicing of other high-risk HPV E6*I/E7 mRNAs, but had no effect on low-risk HPV mRNAs. In summary, we have identified a novel splicing enhancer in the E6 coding region that is specific for high-risk HPVs and that is critically linked to HPV16 carcinogenic properties.

Activating STING/TBK1 suppresses tumor growth via degrading HPV16/18 E7 oncoproteins in cervical cancer.

Cervical cancer is the most common gynecologic cancer, etiologically related to persistent infection of human papillomavirus (HPV). Both the host innate immunity system and the invading HPV have developed sophisticated and effective mechanisms to counteract each other. As a central innate immune sensing signaling adaptor, stimulator of interferon genes (STING) plays a pivotal role in antiviral and antitumor immunity, while viral oncoproteins E7, especially from HPV16/18, are responsible for cell proliferation in cervical cancer, and can inhibit the activity of STING as reported. In this report, we find that activation of STING-TBK1 (TANK-binding kinase 1) promotes the ubiquitin-proteasome degradation of E7 oncoproteins to suppress cervical cancer growth. Mechanistically, TBK1 is able to phosphorylate HPV16/18 E7 oncoproteins at Ser71/Ser78, promoting the ubiquitination and degradation of E7 oncoproteins by E3 ligase HUWE1. Functionally, activated STING inhibits cervical cancer cell proliferation via down-regulating E7 oncoproteins in a TBK1-dependent manner and potentially synergizes with radiation to achieve better effects for antitumor. Furthermore, either genetically or pharmacologically activation of STING-TBK1 suppresses cervical cancer growth in mice, which is independent on its innate immune defense. In conclusion, our findings represent a new layer of the host innate immune defense against oncovirus and provide that activating STING/TBK1 could be a promising strategy to treat patients with HPV-positive cervical cancer.

Human papillomavirus type 16 E6 promotes cervical cancer proliferation by upregulating transketolase enzymatic activity through the activation of protein kinase B.

Over 99% of precancerous cervical lesions are associated with human papillomavirus (HPV) infection, with HPV types 16 and 18 (especially type 16) found in over 70% of cervical cancer cases globally. E6, a critical HPV gene, triggers malignant proliferation by degrading p53; however, this mechanism alone cannot fully explain the oncogenic effects of HPV16 E6. Therefore, we aimed to investigate new targets of HPV oncogenic mechanisms. Our results revealed significant changes in nonoxidative pentose phosphate pathway (PPP) metabolites in HPV16-positive cells. However, the role of nonoxidative PPP in HPV-associated cell transformation and tumor development remained unexplored. In this study, we investigated the impact and mechanisms of HPV16 E6 on cervical cancer proliferation using the HPV-negative cervical cancer cell line (C33A). HPV16 E6 was found to promote cervical cancer cell proliferation both in vitro and in vivo, activating the nonoxidative PPP. Transketolase (TKT), a key enzyme in the nonoxidative PPP, is highly expressed in cervical cancer tissues and associated with poor prognosis. HPV16 E6 promotes cervical cancer cell proliferation by upregulating TKT activity through the activation of AKT. In addition, oxythiamine (OT), a TKT inhibitor, hindered tumor growth, with enhanced effects when combined with cisplatin (DDP). In conclusion, HPV16 E6 promotes cervical cancer proliferation by upregulating TKT activity through the activation of AKT. OT demonstrates the potential to inhibit HPV16-positive cervical cancer growth, and when combined with DDP, could further enhance the tumor-suppressive effect of DDP.

Different immortalized keratinocyte cell lines display distinct capabilities to differentiate and reconstitute an epidermis in vitro.

Dermatological research relies on the availability of suitable models that most accurately reflect the in vivo situation. Primary keratinocytes obtained from skin reduction surgeries are not only limited by availability but have a short lifespan and show donor-specific variations, which hamper the understanding of general mechanisms. The spontaneously immortalized keratinocyte cell line HaCaT displays chromosomal aberrations and is known to differentiate in an abnormal manner. To overcome these issues, we validated different engineered immortalized cell lines created from primary human keratinocytes (NHK) as model systems to study epidermal function. Cell lines either immortalized by the expression of SV40 large T antigen and hTERT (NHK-SV/TERT) or by transduction with HPV E6/E7 (NHK-E6/E7) were analysed for their growth and differentiation behaviour using 2D and 3D culture systems and compared to primary keratinocytes. Both cell lines displayed a robust proliferative behaviour but were still sensitive to contact inhibition. NHK-E6/E7 could be driven into differentiation by Ca2+ switch, while NHK-SV/TERT needed withdrawal from any proliferative signal to initiate a delayed onset of differentiation. In 3D epidermal models both cell lines were able to reconstitute a stratified epidermis and functional epidermal barrier. However, only NHK-E6/E7 showed a degree of epidermal maturation and stratification that was comparable to primary keratinocytes.

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.

Epidermal growth factor receptor-dependent stimulation of differentiation by human papillomavirus type 16 E5.

Human papillomaviruses (HPVs) infect keratinocytes of stratified squamous epithelia, and persistent infection with high-risk HPV types, such as HPV16, may lead to the development of malignancies. HPV evades host immunity in part by linking its gene expression to the host differentiation program, and therefore relies on differentiation to complete its life cycle. Based on previous reports indicating that the HPV16 protein E5 is important in the late stages of the differentiation-dependent life cycle, we found that organotypic cultures harboring HPV16 genomes lacking E5 showed reduced markers of terminal differentiation compared to wild type HPV16-containing cultures. We found that epidermal growth factor receptor (EGFR) levels and activation were increased in an E5-depdendent manner in these tissues, and that EGFR promoted terminal differentiation and expression of the HPV16 L1 gene. These findings suggest a function for E5 in preserving the ability of HPV16 containing keratinocytes to differentiate, thus facilitating the production of new virus progeny.

Genetic and epigenetic alterations in DNA repair genes and treatment outcome of chemoradiotherapy in cervical cancer.

Cervical cancer (CaCx) is the deadliest malignancy among women which is caused by human papillomavirus (HPV) and anthro-demographical/clinicopathological factors. HPV oncoproteins E6 and E7 target p53 and RB (retinoblastoma) protein degradation, Ataxia telangiectasia mutated (ATM), ATM-RAD3-related (ATR) inactivation and subsequent impairment of non-homologous end joining (NHEJ), homologous recombination, and base excision repair pathways. There is also an accumulation of genetic and epigenetic alterations in Tumor Growth Suppressors (TGS), oncogenes, and DNA repair genes leading to increased genome instability and CaCx development. These alterations might be responsible for differential clinical response to Cisplatin-based chemoradiotherapy (CRT) in patients. This review explores HPV-mediated DNA damage as a risk factor in CaCx development, the mechanistic role of genetic and epigenetic alterations in DNA repair genes and their association with CRT and outcome, It also explores new possibilities for the development of genetic and epigenetic-based biomarkers for diagnostic, prognostic, and molecular therapeutic interventions.

The E6 gene polymorphism of Human papillomavirus 16 in relation to the risk of cervical cancer in Tunisian women.

Human papillomavirus type 16 (HPV-16) is the most prevalent HPV type worldwide and in Tunisia and the major carcinogenic HPV type found in cervical precancers and cancers. Previous studies have reported that genetic diversity of HPV16-E6 oncoprotein might be associated with cervical intraepithelial neoplasia progression. In this study we aimed to investigate the prevalence of HPV-16 E6 variants in precancerous lesions in Tunisian population to assess potential correlation with disease severity. Positive HPV cervical samples were obtained from the Laboratory of Anatomy Pathology of Pasteur Institute of Tunis. Cytological study was performed to identify cervical precancerous lesions. HPVs were typed using Reverse Line Hybridization. Only samples with HPV-16 single infection were selected for HP16-E6 genetic diversity investigation. HPV-16 E6 gene amplification was performed by PCR using specific primers and sequenced by Sanger Sequencing. The multiple alignment of generated sequences was performed using MEGAX software. Phylogenetic tree was constructed using Maximum Likehood method. The ternary complex of E6, E6AP and p53 core domain was used to perform in silico point mutations and thermodynamic calculations to assess stability and binding affinity. Genetic analysis of Tunisian E6-HPV16 sequences showed the presence of three lineages: European (A), African (C) and Asian American (D). Interestingly, the EUR variants were identified as the dominant lineage of HPV-16 and HPV-16 E6 350 G (L83V) was the most detected mutation in precancerous lesions. Modelling data showed that African variants induced the largest destabilizing effect on E6 structure and decreasing thereby in the affinity toward E6AP. Therefore, women infected with European variants are associated with low and high intraepithelial lesions. The findings give useful information for personalized decision algorithms of intra-epithelial cervical neoplasia in Tunisian women.

Bioinformatics analysis of immune characteristics in tumors with alternative carcinogenesis pathways induced by human papillomaviruses.

BACKGROUND: Human papillomaviruses (HPVs) induce a subset of head and neck squamous cell carcinomas (HNSCC) and anogenital cancers, particularly cervical cancer (CC). The major viral proteins that contribute to tumorigenesis are the E6 and E7 oncoproteins, whose expression is usually enhanced after the integration of viral DNA into the host genome. Recently, an alternative tumorigenesis pathway has been suggested in approximately half of HNSCC and CC cases associated with HPV infection. This pathway is characterized by extrachromosomal HPV persistence and increased expression of the viral E2, E4, and E5 genes. The E6, E7, E5, and E2 proteins have been shown to modify the expression of numerous cellular immune-related genes. The antitumor immune response is a critical factor in the prognosis of HPV-driven cancers, and its characterization may contribute to the prediction and personalization of the increasingly used cancer immunotherapy. METHODS: We analyzed the immune characteristics of HPV-dependent tumors and their association with carcinogenesis types. Transcriptomic HNSCC and CC datasets from The Cancer Genome Atlas were used for this analysis. RESULTS: Clustering with immune-related genes resulted in two clusters of HPV16-positive squamous cell carcinomas in both tumor types: cluster 1 had higher activation of immune responses, including stimulation of the antigen processing and presentation pathway, which was associated with higher immune cell infiltration and better overall survival, and cluster 2 was characterized by keratinization. In CC, the distribution of tumor samples into clusters 1 and 2 did not depend on the level of E2/E5 expression, but in HNSCC, most E2/E5-high tumors were localized in cluster 1 and E2/E5-low tumors in cluster 2. Further analysis did not reveal any association between the E2/E5 levels and the expression of immune-related genes. CONCLUSIONS: Our results suggest that while the detection of immune responses associated with preserved expression of genes encoding components of antigen processing and presentation machinery in HPV-driven tumors may be markers of better prognosis and an important factor in therapy selection, the type of carcinogenesis does not seem to play a decisive role in the induction of antitumor immunity.

The Inhibitory Effect of KerraTM, KSTM, and MinozaTM on Human Papillomavirus Infection and Cervical Cancer.

Background and Objectives: Cervical cancer is one of the most common types of frequently found cancers in Thailand. One of the causative agents is the infection of the high-risk human papillomavirus (HPV) type 16 and 18. Traditional medicines are rich sources of bioactive compounds which are a valuable source for the development of novel cancer therapies. In this study, the therapeutic effects of 3 traditional medicines, KerraTM, KSTM, and MinozaTM, were studied on HeLa and CaSki cells. Materials and Methods: The effects of KerraTM, KSTM, and MinozaTM on cancer cells were evaluated through cytotoxicity and cell death assays. The infection assay using HPV-16 pseudovirus was also carried out. Results: All traditional medicines efficiently suppressed cell growths of HeLa and CaSki, with KerraTM being the most potent anticancer agent followed by KSTM and MinozaTM. KerraTM at 158 µg/mL and 261 µg/mL significantly increases the percentage inhibition of the HPV-16 pseudovirus infection in a pre-attachment step in a dose-dependent manner, while KSTM at 261 µg/mL efficiently inhibited viral infection in both pre-attachment and adsorption steps. However, KerraTM, KSTM, and MinozaTM at subtoxic concentrations could not reduce the viral E6 mRNA expressions of HPV-16 and HPV-18. Cell death assay by acridine orange/ethidium bromide showed that KerraTM increased population of dead cells in dose-dependent manner in both CaSki and HeLa. The percentage of secondary necrosis in KerraTM-treated CaSki was higher than that of HeLa cells, while the percentage of late apoptotic cells in HeLa was higher than that of CaSki, indicating that HeLa was more susceptible to KerraTM than CaSki. For KSTM and MinozaTM, these extracts at 250 µg/mL promoted autophagy over cell death. At 500 µg/mL, the percentage of dead cells in KerraTM was higher than that of KSTM and MinozaTM. Conclusions: KerraTM is a potent traditional medicine for promoting cancer cell death. KerraTM is possibly useful in the prevention and treatment of cervical cancer. Further investigation will be carried out to gain a better understanding of the biochemical mechanism and the pharmacological activity underlying this effect.