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Background: Human papillomavirus (HPV) is the main risk factor for the development of squamous cell cervical cancer, and E6 oncoprotein and E7 oncoprotein are important components of the viral genome and its oncogenic potential. It is known that different viral variants of HPV16 have different pathology and impact on the development of neoplasia, although few studies have been performed on South American variants. Objective: Therefore, the present study aimed to analyze in silico the genomic diversity of HPV16 in 20 complete genome variants of South America in the National Center for Biotechnology Information (NCBI) database. Methods: We performed a descriptive study to characterize the polymorphic regions of the E6 and E7 genes in HPV16 variants, using software for genomic data and single nucleotide polymorphism (SNP) analysis and others for phylogenetic analysis. Results: The variants analyzed included six SNPs linked to cancer (A131G, G145T, C335T, T350G, C712A, and T732C) and significant variation (798 nucleotide substitutions). Despite this, the variants showed low genetic diversity. Eighteen variants of unclear significance (VUS) were identified, 10 of which were in the coding E6 regions and 8 in the coding E7 regions. The prevalence of lineage D variants is of concern due to their pathology in cervical cancer and requires more research and epidemiological vigilance regarding their prevalence in the population. Conclusion: The data obtained in this study may contribute to future research on South American variants of HPV16, their pathogenicity, and the development of treatments.
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The persistence of the human papillomavirus type 16 (HPV16) infection on the cervical epithelium contributes to the progression of cervical cancer. Studies have demonstrated that HPV16 genetic variants may be associated with different risks of developing cervical cancer. However, the E5 oncoprotein of HPV16, which is related to several cellular mechanisms in the initial phases of the infection and thus contributes to carcinogenesis, is still little studied. Here we investigate the HPV16 E5 oncogene variants to assess the effects of different mutations on the biological function of the E5 protein. We detected and analyzed the HPV16 E5 oncogene polymorphisms and their phylogenetic relationships. After that, we proposed a tertiary structure analysis of the protein variants, preferential codon usage, and functional activity of the HPV16 E5 protein. Intra-type variants were grouped in the lineages A and D using in silico analysis. The mutations in E5 were located in the T-cell epitopes region. We therefore analyzed the interference of the HPV16 E5 protein in the NF-kB pathway. Our results showed that the variants HPV16E5_49PE and HPV16E5_85PE did not increase the potential of the pathway activation capacity. This study provides additional knowledge about the mechanisms of dispersion of the HPV16 E5 variants, providing evidence that these variants may be relevant to the modulation of the NF-κB signaling pathway.
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Breast cancer risk factors include lifestyle, genetic-hormonal influences, and viral infections. Human papillomavirus (HPV), known primarily as the etiological agent of cervical cancer, also appears active in breast carcinogenesis, as evidenced in our study of 56 patients from northeastern Brazil. We assessed the clinical and sociodemographic characteristics, correlating them with various breast cancer tumor types. HPV detection involved amplifying the L1 region, with viral load measured using the E2/E6 ratio and viral activity indicated by E5 oncogene expression. Predominantly, patients over 56 years of age with healthy lifestyles showed a high incidence of invasive ductal carcinoma and triple-negative breast cancer. HPV was detected in 35.7% of cases, mostly HPV16, which is associated with high viral loads (80 copies per cell) and significant E5 expression. These results hint at a possible link between HPV and breast carcinogenesis, necessitating further studies to explore this association and the underlying viral mechanisms.
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Neoplasias da Mama , Infecções por Papillomavirus , Humanos , Brasil/epidemiologia , Feminino , Pessoa de Meia-Idade , Fatores de Risco , Neoplasias da Mama/virologia , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/epidemiologia , Adulto , Idoso , Papillomaviridae , Carga ViralRESUMO
The E6 and E7 oncoproteins of high-risk types of human papillomavirus (HR-HPV) are crucial for the development of cervical cancer (CC). Small interfering RNAs (siRNAs) are explored as novel therapies that silence these oncogenes, but their clinical use is hampered by inefficient delivery systems. Modification (pegylation) with polyethylene glycol (PEG) of liposomal siRNA complexes (siRNA lipoplexes) may improve systemic stability. We studied the effect of siRNA targeting HPV16 E6, delivered via cationic liposomes (lipoplexes), on cellular processes in a cervical carcinoma cell line (CaSki) and its potential therapeutic use. Lipoplexes-PEG-HPV16 E6, composed of DOTAP, Chol, DOPE, and DSPE-PEG2000 were prepared. The results showed that pegylation (5% DSPE-PEG2000) provided stable siRNA protection, with a particle size of 86.42 ± 3.19 nm and a complexation efficiency of over 80%; the siRNA remained stable for 30 days. These lipoplexes significantly reduced HPV16 E6 protein levels and restored p53 protein expression, inhibiting carcinogenic processes such as proliferation by 25.74%, migration (95.7%), and cell invasion (97.8%) at concentrations of 20 nM, 200 nM, and 80 nM, respectively. In conclusion, cationic lipoplexes-PEG-HPV16 E6 show promise as siRNA carriers for silencing HPV16 E6 in CC.
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Human papillomavirus 16 (HPV 16) infection is associated with several types of cancer, such as head and neck, cervical, anal, and penile cancer. Its oncogenic potential is due to the ability of the E6 and E7 oncoproteins to promote alterations associated with cell transformation. HPV 16 E6 and E7 oncoproteins increase metabolic reprogramming, one of the hallmarks of cancer, by increasing the stability of hypoxia-induced factor 1 α (HIF-1α) and consequently increasing the expression levels of their target genes. In this report, by bioinformatic analysis, we show the possible effect of HPV 16 oncoproteins E6 and E7 on metabolic reprogramming in cancer through the E6-E7-PHD2-VHL-CUL2-ELOC-HIF-1α axis. We proposed that E6 and E7 interact with VHL, CUL2, and ELOC in forming the E3 ubiquitin ligase complex that ubiquitinates HIF-1α for degradation via the proteasome. Based on the information found in the databases, it is proposed that E6 interacts with VHL by blocking its interaction with HIF-1α. On the other hand, E7 interacts with CUL2 and ELOC, preventing their binding to VHL and RBX1, respectively. Consequently, HIF-1α is stabilized and binds with HIF-1ß to form the active HIF1 complex that binds to hypoxia response elements (HREs), allowing the expression of genes related to energy metabolism. In addition, we suggest an effect of E6 and E7 at the level of PHD2, VHL, CUL2, and ELOC gene expression. Here, we propose some miRNAs targeting PHD2, VHL, CUL2, and ELOC mRNAs. The effect of E6 and E7 may be the non-hydroxylation and non-ubiquitination of HIF-1α, which may regulate metabolic processes involved in metabolic reprogramming in cancer upon stabilization, non-degradation, and translocation to the nucleus.
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Vitamin D3 or calcitriol (VitD3) has been shown to have anticancer and anti-inflammatory activity in in vitro models and clinical studies. However, its effect on HPV-16-related cancer has been sparsely explored. In this study, we aimed to determine whether monotherapy or combination therapy with cisplatin (CP) reduces tumor growth and affects survival and systemic inflammation. Treatments were administered to C57BL/6 mice with HPV-16-related tumors (TC-1 cells) as follows: (1) placebo (100 µL vehicle, olive oil, orally administered daily); (2) VitD3 (3.75 µg/kg calcitriol orally administered daily); (3) CP (5 mg/kg intraperitoneally, every 7 days); and (4) VitD3+CP. Tumor growth was monitored for 25 days, survival for 60 days, and the neutrophil-to-lymphocyte ratio (NLR) was evaluated on days 1 (baseline), 7, and 14. VitD3+CP showed greater success in reducing tumor volume compared to CP monotherapy (p = 0.041), while no differences were observed between CP and VitD3 monotherapy (p = 0.671). Furthermore, VitD3+CP prolonged survival compared to CP (p = 0.036) and VitD3 (p = 0.007). Additionally, at day 14 the VitD3 and VitD3+CP groups showed significantly lower NLR values than the CP group (p < 0.05, for both comparisons). Vitamin D3 could be a promising adjuvant in the treatment of cervical cancer or solid tumors and deserves further investigation.
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HPV 16 integration is crucial for the onset and progression of premalignant lesions to invasive squamous cell carcinoma (ISCC) because it promotes the amplification of proto-oncogenes and the silencing of tumor suppressor genes; some of these are proteins with PDZ domains involved in homeostasis and cell polarity. Through a bioinformatics approach based on interaction networks, a group of proteins associated with HPV 16 infection, PDZ domains, and direct physical interaction with E6 and related to different hallmarks of cancer were identified. MAGI-1 was selected to evaluate the expression profile and subcellular localization changes in premalignant lesions and ISCC with HPV 16 in an integrated state in cervical cytology; the profile expression of MAGI-1 diminished according to lesion grade. Surprisingly, in cell lines CaSki and SiHa, the protein localization was cytoplasmic and nuclear. In contrast, in histological samples, a change in subcellular localization from the cytoplasm in low-grade squamous intraepithelial lesions (LSIL) to the nucleus in the high-grade squamous intraepithelial lesion (HSIL) was observed; in in situ carcinomas and ISCC, MAGI-1 expression was absent. In conclusion, MAGI-1 expression could be a potential biomarker for distinguishing those cells with normal morphology but with HPV 16 integrated from those showing morphology-related uterine cervical lesions associated with tumor progression.
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BACKGROUND: HPV is strongly related to cervical cancer. HPV lineages can contribute to a response to cervical cancer therapy. The aim of this research was to estimate the frequency of human papillomavirus (HPV)-16 lineages in specimens of cervical cancer, relate the pathological factors in these variants, and assess their response to treatment with radical chemoradiotherapy. METHODS: Samples of cervical cancer were collected from women who were referred to a reference cancer hospital to test the presence of human papillomavirus-type DNA. The standard protocol of this service consisted of cisplatin-based chemotherapy of 40 mg/m2, plus conventional pelvic irradiation in doses of 45-50.4 Gy and high dose-rate brachytherapy of 28-30 Gy to Point A. The response to chemotherapy was evaluated after three months in patients with the HPV-16 lineage. RESULTS: HPV DNA was detected in 104 (88.1%) of the 118 patients. HPV-16 was present in 63 patients (53%). Lineages of HPV-16 were identified in 57 patients and comprised 33 instances of (57.8%) lineage A, 2 instances of lineage B (3.5%), 2 instances of lineage C (3.5%), and 20 instances of (35.0%) lineage D. The median age of the patients was 48.4 years (range 25-85 years). Squamous cell carcinoma was detected 48 times (84.2%). Adenocarcinoma was more likely to occur in lineage D, as three of the four cases occurred in this lineage. A total of 11 patients with the HPV-16 variant were treated with chemoradiotherapy. After three months, it was observed that nine of the eleven patients (81.8%) achieved a complete response, five with the lineage A type, two with the lineage C type, and two with the lineage D type. The two cases of partial response and disease progression, one of each, occurred in lineage A. CONCLUSIONS: In addition to the small number of patients and HPV variants, we noticed a better response in patients with the HPV-16 lineage A. Increasing the sample size could be helpful to better assess the impact of HPV variants on cervical cancer treatment.
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Persistent high-risk human papillomavirus infection is the main risk factor for cervical cancer establishment, where the viral oncogenes E6 and E7 promote a cancerous phenotype. Metabolic reprogramming in cancer involves alterations in glutamine metabolism, also named glutaminolysis, to provide energy for supporting cancer processes including migration, proliferation, and production of reactive oxygen species, among others. The aim of this work was to analyze the effect of HPV16 E6 and E7 oncoproteins on the regulation of glutaminolysis and its contribution to cell proliferation. We found that the E6 and E7 oncoproteins exacerbate cell proliferation in a glutamine-dependent manner. Both oncoproteins increased the levels of transporter SNAT1, as well as GLS2 and GS enzymes; E6 also increased LAT1 transporter protein levels, while E7 increased ASCT2 and xCT. Some of these alterations are also regulated at a transcriptional level. Consistently, the amount of SNAT1 protein decreased in Ca Ski cells when E6 and E7 expression was knocked down. In addition, we demonstrated that cell proliferation was partially dependent on SNAT1 in the presence of glutamine. Interestingly, SNAT1 expression was higher in cervical cancer compared with normal cervical cells. The high expression of SNAT1 was associated with poor overall survival of cervical cancer patients. Our results indicate that HPV oncoproteins exacerbate glutaminolysis supporting the malignant phenotype.
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Glutamina , Neoplasias do Colo do Útero , Feminino , Humanos , Proliferação de Células , Papillomavirus Humano 16/genética , Proteínas E7 de Papillomavirus/genética , Sistema A de Transporte de Aminoácidos/metabolismoRESUMO
PURPOSE: The mechanism of methylation of HPV CpG sites in the occurrence and prognosis of cervical carcinogenesis remains unclear. We investigated the effects of demethylation of the CpG sites of E2 and E6, essential genes of HPV16 integration, on cervical cancer cell expression, integration, and proliferation. MATERIALS AND METHODS: HPV16-positive (Caski) cells were treated with different concentrations of the demethylation compound 5-aza-dc (0, 5, 10, 20 µmol/l) in vitro. After the intervention, the methylation statuses of HPV16 E2 and E6 were detected by TBS, the expression levels of E2 and E6 mRNA and protein were detected by real-time PCR and western blot, cell proliferation activity was detected by CCK8, and cell cycle and apoptosis were determined by FCM. GraphPad Prism version 8.4.2 and R version 4.2.3 were used for relevant data analyses. RESULTS: The methylation levels of HPV16 E2 and E6 CpG sites decreased gradually with increasing 5-aza-dc intervention concentrations. With decreasing E2 and E6 methylation rates, E2 expression increased, the E2/E6 ratio increased, E6 expression decreased, and the growth inhibition rate of Caski cells increased. E2 and E6 expression were negatively and positively correlated with their degrees of methylation respectively, while the E2/E6 mRNA to protein ratio was negatively correlated with the methylation degrees of E2 and E6. CONCLUSION: Demethylation can be used as a prospective treatment to affect HPV expression and persistent infection, providing a new theoretical basis for the clinical treatment of viral infections.
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Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Neoplasias do Colo do Útero , Feminino , Humanos , Neoplasias do Colo do Útero/metabolismo , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Infecções por Papillomavirus/genética , Genes Essenciais , Metilação de DNA , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proliferação de Células , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Abstract Objective Similar to Human Papillomavirus (HPV) genotypes, different lineages of a genotype also have different carcinogenic capabilities. Studies have shown that specific genotype lineages of oncogenic HPV are associated with variable risks for the development of cervical intraepithelial neoplasia (CIN2/CIN3) and cervical cancer. The present study aimed to analyze the genetic diversity of the HPV16 genotype in women with CIN2/CIN3 and cervical cancer, from the northeast region of Brazil. Methods A cross-sectional multicenter study was conducted in the northeast region of Brazil, from 2014 to 2016. This study included 196 cases of HPV16 variants (59 and 137 cases of CIN2/CIN3 and cervical cancer, respectively). The difference of proportion test was used to compare patients with CIN2/CIN3 and cervical cancer, based on the prevalent HPV16 lineage (p < 0.05). Results According to the histopathological diagnosis, the percentage of lineage frequencies revealed a marginal difference in the prevalence of lineage A in CIN2/CIN3, compared with that in cervical cancer (p = 0.053). For lineage D, the proportion was higher in cancer cases (32.8%), than in CIN2/CIN3 cases (16.9%), with p = 0.023. Conclusion HPV16 lineage A was the most frequent lineage in both CIN2/CIN3 and cervical cancer samples, while lineage D was predominant in cervical cancer, suggesting a possible association between HPV16 lineage D and cervical cancer.
Resumo Objetivo Tanto os tipos quanto as linhagens do Papilomavírus Humano (HPV) parecem ter diferentes capacidades carcinogênicas e estão associados a riscos variados para o desenvolvimento de neoplasia intraepitelial cervical (NIC) e câncer de colo do útero. O presente estudo tem como objetivo analisar a diversidade genética do genótipo HPV 16 nos casos de NIC2/NIC3 e câncer de colo de útero em mulheres da região Nordeste do Brasil. Métodos Estudo transversal de base hospitalar realizado na região Nordeste do Brasil no período de 2014 a 2016. A amostra foi composta por 196 casos da variante HPV-16 (59 casos de NIC2/NIC3 e 137 de câncer do colo do útero). O teste de diferença de proporção foi usado para comparar os grupos NIC2/NIC3 e câncer de colo do útero por linhagem viral em relação à prevalência da linhagem HPV-16. Foi considerada significância estatística o valor de p < 0,05. Resultados As frequências de linhagem por diagnóstico histopatológico mostraram diferença limítrofe da linhagem A no grupo NIC2/NIC3 em relação ao grupo câncer de colo de útero (p = 0,053). Por outro lado, em relação à linhagem D, houve uma proporção maior nos casos de câncer (32,8%) quando comparado ao grupo NIC2/NIC3 (16,9%) e esta diferença se mostrou estatisticamente significante (p = 0,023). Conclusão A linhagem A do HPV-16 foi a mais frequente tanto nas amostras CIN2/CIN3 quanto nas amostras de câncer de colo de útero, enquanto a linhagem D predominou no câncer de colo do útero, sugerindo uma possível associação da linhagem D de HPV-16 com câncer de colo de útero.
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Humanos , Feminino , Papillomavirus Humano 16RESUMO
High-risk human papillomavirus (HPV) infection is the main risk factor for cervical cancer (CC) development, where the continuous expression of E6 and E7 oncoproteins maintain the malignant phenotype. In Mexico, around 70% of CC cases are diagnosed in advanced stages, impacting the survival of patients. The aim of this work was to identify biomarkers affected by HPV-16 E6 and E7 oncoproteins that impact the prognosis of CC patients. Expression profiles dependent on E6 and E7 oncoproteins, as well as their relationship with biological processes and cellular signaling pathways, were analyzed in CC cells. A comparison among expression profiles of E6- and E7-expressing cells and that from a CC cohort obtained from The Cancer Genome Atlas (TCGA) demonstrated that the expression of 13 genes impacts the overall survival (OS). A multivariate analysis revealed that the downregulated expression of RIPOR2 was strongly associated with a worse OS. RIPOR2, including its transcriptional variants, were overwhelmingly depleted in E6- and E7-expressing cells. Finally, in a Mexican cohort, it was found that in premalignant cervical lesions, RIPOR2 expression decreases as the lesions progress; meanwhile, decreased RIPOR2 expression was also associated with a worse OS in CC patients.
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Moléculas de Adesão Celular , Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Neoplasias do Colo do Útero , Feminino , Humanos , Papillomavirus Humano 16 , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/genética , Prognóstico , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia , Moléculas de Adesão Celular/genética , Infecções por Papillomavirus/genéticaRESUMO
El cáncer de cuello uterino es el cuarto cáncer más frecuente en mujeres en el mundo y a nivel mundial, el VPH 16 se encuentra presente en aproximadamente el 60% de los casos. A la fecha, las variantes de VPH 16 se clasifican en 4 linajes y 16 sublinajes asociándose algunas variantes con severidad de lesión. La secuenciación de la región LCR y del gen E6 es utilizada para la clasificación de variantes. Por ello, el objetivo fue optimizar 2 PCRs convencionales para detectar la región LCR y una PCR para el gen E6. Para ello, se utilizaron muestras positivas para VPH 16, cebadores específicos para la región LCR y el gen E6. Se probaron las reacciones a diferentes temperaturas de anillamientos. La concentración de MgCl2, dNTP y cebadores fueron determinadas siguiendo las recomendaciones del fabricante de la enzima ADN polimerasa utilizada. Para la amplificación de la región LCR y el gen E6 del VPH 16, se observaron mejores resultados a una temperatura de anillamiento de 57°C y 50°C, respectivamente. La concentración de MgCl2 utilizada en ambas reacciones fue de 1,5mM, la de dNTP 0,2mM y la de cebadores 0,2uM. La optimización de la reacción de PCR permitirá obtener amplicones aptos para secuenciación, a fin de determinar las variantes génicas y posteriormente evaluar funcionalidad y actividad transcripcional que puedan estar relacionadas con la patogénesis cervical.
Cervical cancer is the fourth most common cancer in women in the world and worldwide HPV 16 is present in approximately 60% of cases. To date, HPV 16 variants are classified into 4 lineages and 16 sublineages, with some variants being associated with lesion severity. Sequencing of the LCR region and the E6 gene is used for variant classification. Therefore, the objective was to optimize two conventional PCRs to detect the LCR region and one PCR for the E6 gene. For this purpose, HPV 16 positive samples, specific primers for the LCR region and the E6 gene were used. The reactions were tested at different alignment temperatures. The concentration of MgCl2, dNTP, and primers was determined following the recommendations of the manufacturer of the DNA polymerase enzyme used. For amplification of the LCR region and the HPV 16 E6 gene, the best results were observed at an annealing temperature of 57°C and 50°C, respectively. The concentration of MgCl2 used in both reactions was 1.5mM, dNTP 0.2mM and primers 0.2uM. The present optimization will be used to sequence the amplified products to determine the variants and subsequently evaluate the functionality and transcriptional activity in order to relate it to cervical pathogenesis.
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The E6 oncoprotein of HPV16 variants differentially alters the transcription of the genes involved in migration and non-coding RNAs such as lncRNAs. The role of the lncRNA MINCR in cervical cancer and its relationship with variants of oncogenic HPV remain unknown. Therefore, the objective of this study was to analyze the effect of the E6 oncoprotein of the AA-c variant of HPV16 in cell migration through the MINCR/miR-28-5p/RAP1B axis. To explore the functional role of MINCR in CC, we used an in vitro model of C33-A cells with exogenous expression of the E6 oncoprotein of the AA-c variant of HPV16. Interfering RNAs performed MINCR silencing, and the expression of miR-28-5p and RAP1B mRNA was analyzed by RT-qPCR. We found that C33-A/AA-c cells expressed MINCR 8-fold higher compared to the control cells. There is an inverse correlation between the expression of miR-28-5p and RAP1B in C33-A/AA-c cells. Our results suggest that MINCR might regulate the expression of RAP1B through the inhibition of miR-28-5p in CC cells expressing the E6 oncoprotein of HPV16 AA-c. We report, for the first time, that the MINCR/miR-28-5p/RAP1B axis positively regulates cell migration in CC-derived cells that express the E6 oncoprotein of the AA-c variant of HPV16.
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MicroRNAs , Proteínas Oncogênicas Virais , RNA Longo não Codificante , Neoplasias do Colo do Útero , Proteínas rap de Ligação ao GTP , Linhagem Celular Tumoral , Movimento Celular , Feminino , Papillomavirus Humano 16 , Humanos , MicroRNAs/genética , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , RNA Longo não Codificante/genética , Proteínas Repressoras , Neoplasias do Colo do Útero/genética , Proteínas rap de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: HPV-16 causes approximately 90% of anal canal (AC) cancers worldwide. This study aimed to evaluate the prevalence and persistence of HPV-16 genetic variants in the AC of men from three different countries (Brazil, Mexico and United States) and to further identify sociodemographic and behavioral factors associated with these infections. METHODS: Participants from the multinational prospective HPV Infection in Men (HIM) Study who had at least one HPV-16 positive AC swab were included. Characterization into HPV-16 genetic variants was successfully performed by PCR-sequencing in 95.6% (217/227) samples and these were classified into HPV-16 lineages and sublineages. RESULTS: We observed higher prevalence of lineage A variants, mainly from A1 sublineage, in all countries. Non-A lineage variants were mostly detected in men from Brazil, where higher diversity of sublineage variants was detected during follow-up. Compare to men detected with Non-A HPV-16 lineage variants, men infected with lineage A reported a higher lifetime number of female sexual partners. Finally, a significantly higher prevalence of Non-A lineage variants was observed among men who have sex with men (MSM) with a transient HPV-16 AC infection (p = 0.033), but no significant differences regarding variants lineages and persistence status were observed when stratified by country, self-reported ethnicity or age. CONCLUSIONS: Our data extend previous reports which indicate that globally HPV-16 variants are unevenly distributed, and contribute further to studies of the natural history of AC HPV infections in men.
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Doenças do Ânus , Infecções por Papillomavirus , Minorias Sexuais e de Gênero , Canal Anal , Doenças do Ânus/epidemiologia , Feminino , Homossexualidade Masculina , Papillomavirus Humano 16/genética , Humanos , Masculino , Papillomaviridae/genética , Infecções por Papillomavirus/epidemiologia , Prevalência , Estudos Prospectivos , Fatores de Risco , Estados UnidosRESUMO
Cervical cancer (CC) is the most common cancer in women in the lower genital tract. The main risk factor for developing CC is persistent infection with HPV 16. The E6 and E7 oncoproteins of HPV 16 have been related to metabolic reprogramming in cancer through the regulation of the expression and stability of HIF-1α and consequently of the expression of its target genes, such as HIF1A (HIF-1α), SLC2A1 (GLUT1), LDHA, CA9 (CAIX), SLC16A3 (MCT4), and BSG (Basigin or CD147), which are involved in glucose metabolism. This work aimed to evaluate the expression of HIF-1α, GLUT1, LDHA, CAIX, MCT4, and Basigin in patient samples and CC cell lines. To evaluate the expression level of HIF1A, SLC2A1, LDHA, CA9, SLC16A3, and BSG genes in tissue from patients with CC and normal tissue, the TCGA dataset was used. To evaluate the expression level of these genes by RT-qPCR in CC cell lines, HPV-negative (C-33A) and HPV-16-positive (SiHa and Ca Ski) cell lines were used. Increased expression of HIF1A, SLC2A1, LDHA, SLC16A3, and BSG was found in Ca Ski and CA9 in SiHa compared to C-33A. Similar results were observed in CC tissues compared to normal tissue obtained by bioinformatics analysis. In conclusion, the expression of HIF-1α, GLUT1, LDHA, CAIX, MCT4, and BSG genes is increased in CC and HPV-16-positive cell lines.
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BACKGROUND: It has been reported that the oncoprotein E7 from human papillomavirus type 16 (HPV16-E7) can induce the excessive synthesis of centrosomes through the increase in the expression of PLK4, which is a transcriptional target of E2F1. On the other hand, it has been reported that increasing MPS1 protein stability can also generate an excessive synthesis of centrosomes. In this work, we analyzed the possible role of MPS1 in the amplification of centrosomes mediated by HPV16-E7. RESULTS: Employing qRT-PCR, Western Blot, and Immunofluorescence techniques, we found that E7 induces an increase in the MPS1 transcript and protein levels in the U2OS cell line, as well as protein stabilization. Besides, we observed that inhibiting the expression of MPS1 in E7 protein-expressing cells leads to a significant reduction in the number of centrosomes. CONCLUSIONS: These results indicate that the presence of the MPS1 protein is necessary for E7 protein to increase the number of centrosomes, and possible implications are discussed.
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Although active immunotherapies are effective strategies to induce activation of CD8+ T cells, advanced stage tumors require further improvements for efficient control. Concerning the burden of cancer-related to Human papillomavirus (HPV), particularly the high incidence and mortality of cervical cancer, our group developed an approach based on a DNA vaccine targeting the HPV-16 E7 oncoprotein (pgDE7h). This immunotherapy is capable of inducing an antitumour CD8+ T cell response but show only partial control of tumors in more advanced growth stages. Here, we combined a chemotherapeutic agent (gemcitabine- Gem) with pgDE7h to overcome immunosuppression and improve antitumour responses in a preclinical mouse tumor model. Our results demonstrated that administration of Gem had synergistic antitumor effects when combined with pgDE7h leading to eradication of both early-stages and established tumors. Overall, the antiproliferative effects of Gem observed in vitro and in vivo provided an optimal window for immunotherapy. In addition, the enhanced antitumour responses induced by the combined therapeutic regimen included enhanced frequencies of antigen-presenting cells (APCs), E7-specific IFN-γ-producing CD8+ T cells, and cytotoxic CD8+ T cells and, concomitantly, less pronounced accumulation of immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). These findings demonstrated that the combination of Gem and an active immunotherapy strategy show increased effectiveness, leading to a reduced need for multiple drug doses and, therefore, decreased deleterious side effects avoiding resistance and tumor relapses. Altogether, our results provide evidence for a new and feasible chemoimmunotherapeutic strategy that supports future clinical translation.
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Alphapapillomavirus , Infecções por Papillomavirus , Vacinas contra Papillomavirus , Neoplasias do Colo do Útero , Vacinas de DNA , Animais , Linfócitos T CD8-Positivos , Desoxicitidina/análogos & derivados , Feminino , Humanos , Camundongos , Papillomaviridae , Infecções por Papillomavirus/tratamento farmacológico , Neoplasias do Colo do Útero/tratamento farmacológico , GencitabinaRESUMO
Persistent infections with some types of human papillomavirus (HPV) constitute the major etiological factor for cervical cancer development. Nanog, a stem cell transcription factor has been shown to increase during cancer progression. We wanted to determine whether Nanog could modulate transcription of E6 and E7 oncogenes. We used luciferase reporters under the regulation of the long control region (LCR) of HPV types 16 and 18 (HPV16/18) and performed RT-qPCR. We found that Nanog increases activity of both viral regulatory regions and elevates endogenous E6/E7 mRNA levels in cervical cancer-derived cells. We demonstrated by in vitro mutagenesis that changes at Nanog-binding sites found in the HPV18 LCR significantly inhibit transcriptional activation. Chromatin immunoprecipitation (ChIP) assays showed that Nanog binds in vivo to the HPV18 LCR, and its overexpression increases its binding as well as that of c-Jun. Surprisingly, we observed that mutation of AP1-binding sites also affect Nanog's ability to activate transcription, suggesting cooperation between the two factors. We searched for putative Nanog-binding sites in the LCR of several HPVs and surprisingly found them only in those types associated with cancer development. Our study shows, for the first time, a role for Nanog in the regulation of E6/E7 transcription of HPV16/18.
Assuntos
Proteínas de Ligação a DNA/genética , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Proteína Homeobox Nanog/metabolismo , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/metabolismo , Proteínas Repressoras/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 18/metabolismo , Humanos , Proteína Homeobox Nanog/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologiaRESUMO
In this work, a low-cost and rapid electrochemical resistive DNA biosensor based on the current relaxation method is described. A DNA probe, complementary to the specific human papillomavirus type 16 (HPV-16) sequence, was immobilized onto a screen-printed gold electrode. DNA hybridization was detected by applying a potential step of 30 mV to the system, composed of an external capacitor and the modified electrode DNA/gold, for 750 µs and then relaxed back to the OCP, at which point the voltage and current discharging curves are registered for 25 ms. From the discharging curves, the potential and current relaxation were evaluated, and by using Ohm's law, the charge transfer resistance through the DNA-modified electrode was calculated. The presence of a complementary sequence was detected by the change in resistance when the ssDNA is transformed in dsDNA due to the hybridization event. The target DNA concentration was detected in the range of 5 to 20 nM. The results showed a good fit to the regression equation ΔRtotal(Ω)=2.99 × [DNA]+81.55, and a detection limit of 2.39 nM was obtained. As the sensing approach uses a direct current, the electronic architecture of the biosensor is simple and allows for the separation of faradic and nonfaradaic contributions. The simple electrochemical resistive biosensor reported here is a good candidate for the point-of-care diagnosis of HPV at a low cost and in a short detection time.