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1.
Mem Inst Oswaldo Cruz ; 115: e190405, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32187327

RESUMO

BACKGROUND: High-risk human papillomaviruses (HR-HPVs) are the etiological agents of cervical cancer. Among them, types 16 and 18 are the most prevalent worldwide. The HPV genome encodes three oncoproteins (E5, E6, and E7) that possess a high transformation potential in culture cells when transduced simultaneously. In the present study, we analysed how these oncoproteins cooperate to boost key cancer cell features such as uncontrolled cell proliferation, invasion potential, and cellular redox state imbalance. Oxidative stress is known to contribute to the carcinogenic process, as reactive oxygen species (ROS) constitute a potentially harmful by-product of many cellular reactions, and an efficient clearance mechanism is therefore required. Cells infected with HR-HPVs can adapt to oxidative stress conditions by upregulating the formation of endogenous antioxidants such as catalase, glutathione (GSH), and peroxiredoxin (PRX). OBJECTIVES: The primary aim of this work was to study how these oncoproteins cooperate to promote the development of certain cancer cell features such as uncontrolled cell proliferation, invasion potential, and oxidative stress that are known to aid in the carcinogenic process. METHODS: To perform this study, we generated three different HaCaT cell lines using retroviral transduction that stably expressed combinations of HPV-18 oncogenes that included HaCaT E5-18, HaCaT E6/E7-18, and HaCaT E5/E6/E7-18. FINDINGS: Our results revealed a statistically significant increment in cell viability as measured by MTT assay, cell proliferation, and invasion assays in the cell line containing the three viral oncogenes. Additionally, we observed that cells expressing HPV-18 E5/E6/E7 exhibited a decrease in catalase activity and a significant augmentation of GSH and PRX1 levels relative to those of E5, E6/E7, and HaCaT cells. MAIN CONCLUSIONS: This study demonstrates for the first time that HPV-18 E5, E6, and E7 oncoproteins can cooperate to enhance malignant transformation.


Assuntos
Transformação Celular Viral/genética , Proteínas de Ligação a DNA/metabolismo , Papillomavirus Humano 18/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Linhagem Celular Tumoral/virologia , Proliferação de Células , Sobrevivência Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Oxirredução
2.
Int J Oral Sci ; 12(1): 3, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911577

RESUMO

High-risk human papillomaviruses (HPVs) are involved in the development of several human cancers, including oropharyngeal squamous cell carcinomas. However, many studies have demonstrated that HPV alone is not sufficient for the oncogenic transformation of normal human epithelial cells, indicating that additional cofactors are required for the oncogenic conversion of HPV-infected cells. Inasmuch as chronic inflammation is also closely associated with carcinogenesis, we investigated the effect of chronic exposure to tumor necrosis factor α (TNFα), the major proinflammatory cytokine, on oncogenesis in two immortalized oral keratinocyte cell lines, namely, HPV16-immortalized and human telomerase reverse transcriptase (hTERT)-immortalized cells. TNFα treatment led to the acquisition of malignant growth properties in HPV16-immortalized cells, such as (1) calcium resistance, (2) anchorage independence, and (3) increased cell proliferation in vivo. Moreover, TNFα increased the cancer stem cell-like population and stemness phenotype in HPV16-immortalized cells. However, such transforming effects were not observed in hTERT-immortalized cells, suggesting an HPV-specific role in TNFα-promoted oncogenesis. We also generated hTERT-immortalized cells that express HPV16 E6 and E7. Chronic TNFα exposure successfully induced the malignant growth and stemness phenotype in the E6-expressing cells but not in the control and E7-expressing cells. We further demonstrated that HPV16 E6 played a key role in TNFα-induced cancer stemness via suppression of the stemness-inhibiting microRNAs miR-203 and miR-200c. Overexpression of miR-203 and miR-200c suppressed cancer stemness in TNFα-treated HPV16-immortalized cells. Overall, our study suggests that chronic inflammation promotes cancer stemness in HPV-infected cells, thereby promoting HPV-associated oral carcinogenesis.


Assuntos
Carcinoma de Células Escamosas/genética , Papillomavirus Humano 16/metabolismo , MicroRNAs/metabolismo , Neoplasias Bucais/genética , Boca/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/virologia , Telomerase/genética , Fator de Necrose Tumoral alfa/metabolismo , Carcinogênese/genética , Carcinogênese/imunologia , Carcinoma de Células Escamosas/patologia , Transformação Celular Viral/genética , Regulação da Expressão Gênica , Genes Virais , Papillomavirus Humano 16/genética , Humanos , MicroRNAs/genética , Boca/virologia , Neoplasias Bucais/patologia , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Telomerase/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
3.
PLoS Pathog ; 16(1): e1008295, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31971989

RESUMO

The HECT domain E3 ubiquitin ligase E6AP (UBE3A) is critical for the development of human papillomavirus (HPV) associated cancers, the neurodevelopment disorder Angelman Syndrome, and some cases of autism spectrum disorders. How E6AP recognizes its cellular targets and how its ubiquitin ligase activity is triggered remain poorly understood, and HPV E6 proteins are models for these processes. We examined diverse E6 proteins from human and non-human papillomaviruses and identified two different modes of interaction between E6 and E6AP. In Type I interactions, E6 can interact directly with the LXXLL peptide motif alone of E6AP (isolated from the rest of E6AP), and then recruit cellular substrates such as p53. In Type II interactions, E6 proteins require additional auxiliary regions of E6AP in either the amino terminus or in the carboxy-terminal HECT domain to interact with the LXXLL peptide motif of E6AP. A region of E6AP amino-terminal to the LXXLL peptide motif both augments association with E6 proteins and is required for E6 proteins to trigger ubiquitin ligase activity in the carboxy-terminal HECT ubiquitin ligase domain of E6AP. In Type I interactions, E6 can associate with E6AP and recruit p53, but a Type II interaction is required for the degradation of p53 or NHERF1. Interestingly, different E6 proteins varied in E6AP auxiliary regions that contributed to enhanced association, indicating evolutionary drift in the formation of Type II interactions. This classification of E6-E6AP interaction types and identification of a region in the E6AP amino terminus that is important for both E6 association and stimulation of ubiquitin ligase activity will inform future structural data of the E6-E6AP complex and future studies aiming to interfere with the activity of the E6-E6AP complex.


Assuntos
Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/metabolismo , Infecções por Papillomavirus/enzimologia , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Humanos , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética
4.
Int J Cancer ; 146(2): 461-474, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31603527

RESUMO

The malignant growth of human papillomavirus (HPV)-positive cancer cells is dependent on the continuous expression of the viral E6/E7 oncogenes. Here, we examined the effects of iron deprivation on the phenotype of HPV-positive cervical cancer cells. We found that iron chelators, such as the topical antifungal agent ciclopirox (CPX), strongly repress HPV E6/E7 oncogene expression, both at the transcript and protein level. CPX efficiently blocks the proliferation of HPV-positive cancer cells by inducing cellular senescence. Although active mTOR signaling is considered to be critical for the cellular senescence response towards a variety of prosenescent agents, CPX-induced senescence occurs under conditions of severely impaired mTOR signaling. Prolonged CPX treatment leads to p53-independent Caspase-3/7 activation and induction of apoptosis. CPX also eliminates HPV-positive cancer cells under hypoxic conditions through induction of apoptosis. Taken together, these results show that iron deprivation exerts profound antiviral and antiproliferative effects in HPV-positive cancer cells and suggest that iron chelators, such as CPX, possess therapeutic potential as HPV-inhibitory, prosenescent and proapoptotic agents in both normoxic and hypoxic environments.


Assuntos
Ciclopirox/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas Oncogênicas Virais/antagonistas & inibidores , Proteínas E7 de Papillomavirus/antagonistas & inibidores , Infecções por Papillomavirus/tratamento farmacológico , Proteínas Repressoras/antagonistas & inibidores , Neoplasias do Colo do Útero/tratamento farmacológico , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Apoptose/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Ciclopirox/uso terapêutico , Proteínas de Ligação a DNA/metabolismo , Feminino , Células HCT116 , Células HeLa , Humanos , Quelantes de Ferro/farmacologia , Quelantes de Ferro/uso terapêutico , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Proteínas Repressoras/metabolismo , Esferoides Celulares , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia
5.
Dokl Biochem Biophys ; 488(1): 296-299, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31768844

RESUMO

The antiproliferative effect of the "early" protein E2 of the high-risk oncogenic human papillomavirus HPV16 on mouse testis tumors, which were induced by the intramuscular injection of HeLa cells, was discovered. The regression of tumors was maximum in the first 2 days after the oral vaccination with HPV16 E2 (500 mg per mouse) and then gradually decreased to the control variant. A typical monolayer of HeLa cells on the cultivation flask bottom, of which only 18% were functionally active, appeared after seeding testis tissue cells on DMEM medium.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Papillomavirus Humano 16 , Neoplasias Experimentais/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Neoplasias Testiculares/metabolismo , Animais , Proliferação de Células , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Masculino , Camundongos , Neoplasias Experimentais/genética , Proteínas Oncogênicas Virais/genética , Neoplasias Testiculares/genética
6.
Pathog Dis ; 77(5)2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31504464

RESUMO

Human papillomavirus (HPV) associated cancers, and in particular cervical cancer, are considered to be directly stimulated by HPV oncogenes. Alternatively, these types of cancers could also be indirectly stimulated by HPV-induced chronic inflammations, which in turn are also caused by HPV oncogenes activity. Chronic inflammation is associated with repeated tissue injury and development of mutations in the vital tumor suppressor genes. Thus, it is important to understand that the persistent HPV infection and its associated chronic inflammation is responsible for the progression of HPV-induced cancers. HPV E5, E6 and E7 could upregulate the expression of cyclooxygenase (COX)-2 and prostaglandin (PG) E2 followed by the activation of the COX-PG pathway. This pathway is assumed to be the main cause of HPV-induced inflammation. Additionally, HPV oncogenes could have an impact on the upregulation of pro-inflammatory cytokines in HPV-positive patients. The upregulation of such cytokines accelerates the incidence of inflammation following HPV infection. Other factors such as microRNAs, which are involved in the inflammation pathways and aging, give rise to the increased level of pro-inflammatory cytokines and could also be responsible for the acceleration of HPV-induced inflammation and consequent cervical cancer. In this review, the exact roles of HPV oncogenes in the occurrence of inflammation in cervical tissue, and the effects of other factors in this event are evaluated.


Assuntos
Interações Hospedeiro-Patógeno , Inflamação/complicações , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/complicações , Neoplasias do Colo do Útero/etiologia , Doença Crônica , Feminino , Humanos , Papillomaviridae/crescimento & desenvolvimento , Papillomaviridae/patogenicidade
7.
Gynecol Oncol ; 155(2): 340-348, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31477279

RESUMO

OBJECTIVE: To determine the involvement of homeobox D9 (HOXD9) in the survival, proliferation, and metastasis of cervical cancer cells through regulating the expression of human papillomavirus (HPV) 16 E6/E7 genes using the P97 promoter. METHODS: One hundred cases of cervical cancer (CC), CC cell lines SKG-I, SKG-II, SKG-IIIa, SKG-IIIb, HeLa, and SiHa, and a human tumor xenograft mouse model were used to examine the roles of HOXD9 in CC. Knockdown experiments employed RNA interference of HOXD9. qPCR, functional assays, western blotting, DNA microarray, and luciferase and ChIP assays were applied for assessments. RESULTS: All CC cell lines expressed HOXD9 mRNA and protein. In uterine CC, HOXD9 gene expression was significantly higher than in normal cervical tissues. A positive correlation of lymphovascular space invasion and lymph node metastasis with high levels of HOXD9 expression was found in patient samples. HOXD9-knockdown cells in the mouse xenograft model only formed small or no tumors. Knockdown of HOXD9 markedly reduced CC cell proliferation, migration and invasion, induced apoptosis, increased P53 protein expression, and suppressed HPV E6/E7 expression by directly binding to the P97 promoter of HPV16 E6/E7 genes. A positive correlation between HOXD9 and HPV16 E6 expression was found in CC patients. CONCLUSIONS: HOXD9 promotes HPV16 E6 and E7 expression by direct binding to the P97 promoter, which enhances proliferation, migration, and metastasis of CCr cells. Our results suggest that HOXD9 could be a prognostic biomarker and potential therapeutic target in CC.


Assuntos
Proteínas de Homeodomínio/fisiologia , Proteínas de Neoplasias/fisiologia , Infecções por Papillomavirus/genética , Regiões Promotoras Genéticas/genética , Neoplasias do Colo do Útero/virologia , Animais , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Papillomavirus Humano 16/genética , Humanos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Proteínas Oncogênicas Virais/metabolismo , Oncogenes , Proteínas E7 de Papillomavirus/metabolismo , Fenótipo , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/genética
8.
Int J Med Sci ; 16(8): 1096-1101, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31523171

RESUMO

Objective: Efficient and highly predictive biomarkers reflecting the prognosis of persistent atypical squamous cells of unknown significance(ASCUS) and low grade squamous intraepithelial lesion(LSIL)s are unavailable and need to be developed urgently. We aimed to develop a predictive model for diagnosis of cervical intraepithelial neoplasia(CIN)2+ by analyzing the immunocytochemical expression of the HPV L1 capsid protein in patients with persistent ASCUS and LSIL with a high risk of HPV infection. Methods: Cervical cytology samples comprising (70 ASCUS and 215 LSIL Pap smears) were analyzed. Immunocytochemical identification of the HPV L1 capsid protein in cervical cytology samples was performed. Expression levels of HPV L1 capsid protein in cervical cytology samples were measured, and the correlation between HPV L1 expression and cervical pathologic diagnosis was evaluated. The risk for CIN2+ was calculated using the results of immunocytochemistry and the HPV DNA test. Results: Negative results for HPV L1 immunochemistry test were more frequently observed in CIN2+, and expression of the HPV L1 capsid protein was higher in CIN1 or cervicitis (Fisher's exact test, p<0.05). Diagnosis rates for CIN2+ were highest for the combination of HPV L1 capsid protein immunocytochemistry, cytology and HPV test when compared with other combinations (Akaike information criterion (AIC): 191.7, Schwarz criterion(SC): 206.3, p<0.001). Conclusion: Absence of HPV L1 capsid expression and presence of HPV type 16 or 18 infection are reliable predictors of progression to CIN2+ in patients showing persistent ASCUS and LSIL.


Assuntos
Células Escamosas Atípicas do Colo do Útero/virologia , Biomarcadores Tumorais/análise , Proteínas do Capsídeo/análise , Neoplasia Intraepitelial Cervical/virologia , Proteínas Oncogênicas Virais/análise , Infecções por Papillomavirus/patologia , Neoplasias do Colo do Útero/virologia , Adulto , Idoso , Células Escamosas Atípicas do Colo do Útero/metabolismo , Células Escamosas Atípicas do Colo do Útero/patologia , Proteínas do Capsídeo/metabolismo , Neoplasia Intraepitelial Cervical/metabolismo , Neoplasia Intraepitelial Cervical/patologia , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/virologia , Prognóstico , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Esfregaço Vaginal , Adulto Jovem
9.
Arch Virol ; 164(12): 2953-2961, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31552532

RESUMO

Human papillomavirus genotype 16 (HPV16) is the most frequent high-risk HPV (HR-HPV) identified in cervical precursor lesions and cervical cancer (CC) worldwide. The oncogenic potential of HPV16 is partly dependent on the lineage involved in the infection and the presence of clinically relevant mutations. In this report, we present the distribution of HR-HPV and the mutational profile and intra-host variability of HPV16 lineages, based on analysis of the long control region (LCR) and the E6 gene in samples with normal cytology (n = 39), squamous intraepithelial lesions (n = 25), and CC (n = 39). HR-HPV genotyping was performed using multiplex real-time PCR. HPV16 lineage assignments and mutation frequencies were determined by conventional PCR and Sanger DNA sequencing, and intra-patient viral populations were analyzed using next-generation sequencing (NGS). The most frequent HR-HPV type was HPV16, followed by HPV31 and HPV18. The frequency of HPV16 sublineages was A1/A2 > D2 > D3 and B1. Moreover, the most frequent mutations, both in samples from this study and in the available sequences from Mexican isolates in the GenBank database were LCR-G7518A, which is involved in carcinogenesis, and E6-T350G (producing L83V), associated with persistence of infection. Otherwise, deep sequencing revealed high conservation of viral lineages and mutations, independently of the stages studied. In conclusion, the high frequency and stability of these molecular markers, as well as the circulating viral lineages, could be related to the incidence of CC associated with HPV16. Hence, they deserve a broader analysis to determine the risk of specific populations for progression of the disease.


Assuntos
Papillomavirus Humano 16/genética , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/virologia , Proteínas Repressoras/genética , Sequências Repetidas Terminais , Neoplasias do Colo do Útero/virologia , Adulto , Sequência de Bases , Feminino , Regulação Viral da Expressão Gênica , Papillomavirus Humano 16/classificação , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 16/metabolismo , Humanos , México , Mutação , Proteínas Oncogênicas Virais/metabolismo , Filogenia , Proteínas Repressoras/metabolismo , Estudos Retrospectivos
10.
Oncol Rep ; 42(5): 2139-2148, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31436299

RESUMO

Curcumin is a natural antioxidant polyphenol, which decreases epithelial­mesenchymal transition (EMT) and cell migration in cervical cancer cells. However, the mechanism by which such a decrease occurs is unclear. It is well established that cervical cancer can be caused by high­risk human papillomavirus (HPV), which overexpresses E6 and E7 oncoproteins. Recent findings have suggested that viral oncoproteins regulate the expression of Pirin, which is an oxidative stress sensor involved in EMT and cell migration. Molecular markers associated with EMT, pirin and HPV were evaluated using reverse transcription­reverse quantitative PCR and western blotting. In addition, the migratory ability of cells was evaluated using a Transwell assay. In order to evaluate the role of Pirin in curcumin­mediated inhibition of EMT, SiHa cervical carcinoma cells, which contain two integrated copies of HPV16, were exposed to curcumin. Cell migration, and the expression levels of EMT biomarkers and the pirin protein, which is a product of the PIR gene, were subsequently evaluated. The results demonstrated a significant decrease in EMT following exposure to 20 µM curcumin for 72 h. This finding was supported by a decrease in the protein expression levels of N­cadherin, Vimentin and Slug. Furthermore, it was observed that PIR expression and Pirin protein levels were significantly decreased when SiHa cells were exposed to curcumin. Subsequently, to analyze the effects of Pirin on EMT, SiHa cells were transfected with a small interfering RNA (siRNA) to knockdown PIR. A significant increase in E­cadherin mRNA expression and a decrease in N­cadherin protein expression were observed. In addition, a similar decrease was observed when SiHa cells were exposed to both PIR siRNA and curcumin. Finally, a significant decrease in SiHa cell migration was observed in the presence of 20 µM curcumin compared with in the control group. These findings suggested that curcumin may decrease EMT, at least in part by a Pirin­dependent mechanism. Therefore, Pirin protein may be an important pharmacological target for cervical cancer treatment.


Assuntos
Antineoplásicos/farmacologia , Curcumina/farmacologia , Dioxigenases/genética , Dioxigenases/metabolismo , Neoplasias do Colo do Útero/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Ensaios de Seleção de Medicamentos Antitumorais , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Humanos , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia
11.
Proc Natl Acad Sci U S A ; 116(35): 17470-17479, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31395738

RESUMO

The most frequently mutated protein in human cancer is p53, a transcription factor (TF) that regulates myriad genes instrumental in diverse cellular outcomes including growth arrest and cell death. Cell context-dependent p53 modulation is critical for this life-or-death balance, yet remains incompletely understood. Here we identify sequence signatures enriched in genomic p53-binding sites modulated by the transcription cofactor iASPP. Moreover, our p53-iASPP crystal structure reveals that iASPP displaces the p53 L1 loop-which mediates sequence-specific interactions with the signature-corresponding base-without perturbing other DNA-recognizing modules of the p53 DNA-binding domain. A TF commonly uses multiple structural modules to recognize its cognate DNA, and thus this mechanism of a cofactor fine-tuning TF-DNA interactions through targeting a particular module is likely widespread. Previously, all tumor suppressors and oncoproteins that associate with the p53 DNA-binding domain-except the oncogenic E6 from human papillomaviruses (HPVs)-structurally cluster at the DNA-binding site of p53, complicating drug design. By contrast, iASPP inhibits p53 through a distinct surface overlapping the E6 footprint, opening prospects for p53-targeting precision medicine to improve cancer therapy.


Assuntos
DNA/genética , DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Repressoras/metabolismo , Elementos de Resposta , Proteína Supressora de Tumor p53/metabolismo , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , DNA/química , Perfilação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Modelos Moleculares , Motivos de Nucleotídeos , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Repressoras/química , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/química
12.
BMC Mol Cell Biol ; 20(1): 30, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387520

RESUMO

BACKGROUND: Several human cancers, especially cervical cancer are caused by the infection of high risk strains of human papillomaviruses (HPV), notably HPV16. It is implicated that the oncoprotein E6 expressed from HPV, is inhibiting the apoptotic pathway by binding to adaptor molecule FADD (Fas-associated death domain). Inhibiting E6 interactions with FADD could provide a promising treatment for cervical cancer. There are few small molecules reported to inhibit such interactions. However, the FADD binding site information on the HPV E6 is not currently available. This binding site information may provide an opportunity to design new small molecule inhibitors to treat E6 mediated cancers. In this study we report the possible binding pocket on HPV16 E6 oncoprotein by using activity data of reported inhibitors through a stepwise molecular modeling approach. RESULTS: Blind docking and removing duplicates followed by visual inspection to determine ligand-receptor interactions provided 68 possible binding sites on the E6 protein. Individual docking of all known inhibitors lead to the identification of 28 pockets having some kind of correlation with their activity data. It was also observed that several of these pockets overlapped with each other, having some amino acids in common. Amino acids Leu50 and Cys51 were identified as key E6 residues for high affinity ligand binding which are seen in most of these pockets. In most cases, ligands demonstrated a hydrogen bond interaction with Cys51. Ala61, Arg131 and Gln107 were also frequently observed showing interactions among these pockets. A few amino acids unique to each ligand were also identified representing additional interactions at the receptor site. CONCLUSIONS: After determining receptor-ligand interactions between E6 oncoprotein and the six known inhibitors, the amino acids Cys51, Leu50, Arg102, Arg131, Leu67, Val62, and Gln107 were identified to have importance in E6 inhibition. It was generally observed that Leu50 and Cys51 are necessary for high binding affinity with Cys51 being essential for hydrogen bonding. This study identified a potential binding pocket for the E6 inhibitors. Identification of the ligand binding pocket helps to design novel inhibitors of HPV16 E6 oncoprotein as a promising treatment for cervical cancer.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Proteínas Oncogênicas Virais/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Aminoácidos/metabolismo , Sítios de Ligação , Flavonóis/química , Flavonóis/farmacologia , Concentração Inibidora 50 , Ligantes , Simulação de Acoplamento Molecular , Proteínas Oncogênicas/química , Proteínas Oncogênicas Virais/química , Proteínas Repressoras/química
13.
mBio ; 10(4)2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387913

RESUMO

The amino (N)-terminal region of human papillomavirus (HPV) minor capsid protein (L2) is a highly conserved region which is essential for establishing viral infection. Despite its importance in viral infectivity, the role of the HPV N-terminal domain has yet to be fully characterized. Using fine mapping analysis, we identified a 36-amino-acid (aa) peptide sequence of the L2 N terminus, termed L2N, that is critical for HPV infection. Ectopic expression of L2N with the transmembrane sequence on the target cell surface conferred resistance to HPV infection. Additionally, L2N peptide with chemical or enzymatic lipidation at the carboxyl (C) terminus efficiently abrogated HPV infection in target cells. Among the synthetic L2N lipopeptides, a stearoylated lipopeptide spanning aa 13 to 46 (13-46st) exhibited the most potent anti-HPV activity, with a half-maximal inhibitory concentration (IC50) of ∼200 pM. Furthermore, we demonstrated that the 13-46st lipopeptide inhibited HPV entry by blocking trans-Golgi network retrograde trafficking of virion particles, leading to rapid degradation. Fundamentally, the inhibitory effect of L2N lipopeptides appeared to be evolutionarily conserved, as they showed cross-type inhibition among various papillomaviruses. In conclusion, our findings provide new insights into the critical role of the L2N sequence in the HPV entry mechanism and identify the therapeutic potential of L2N lipopeptide as an effective anti-HPV agent.IMPORTANCE HPV is a human oncogenic virus that causes a major public health problem worldwide, which is responsible for approximately 5% of total human cancers and almost all cases of cervical cancers. HPV capsid consists of two structure proteins, the major capsid L1 protein and the minor capsid L2 protein. While L2 plays critical roles during the viral life cycle, the molecular mechanism in viral entry remains elusive. Here, we performed fine mapping of the L2 N-terminal region and defined a short 36-amino-acid peptide, called L2N, which is critical for HPV infection. Specifically, L2N peptide with carboxyl-terminal lipidation acted as a potent and cross-type HPV inhibitor. Taken together, data from our study highlight the essential role of the L2N sequence at the early step of HPV entry and suggests the L2N lipopeptide as a new strategy to broadly prevent HPV infection.


Assuntos
Proteínas do Capsídeo/antagonistas & inibidores , Capsídeo/metabolismo , Papillomavirus Humano 16/efeitos dos fármacos , Lipopeptídeos/farmacologia , Proteínas Oncogênicas Virais/antagonistas & inibidores , Infecções por Papillomavirus/virologia , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/fisiologia , Humanos , Lipopeptídeos/genética , Lipopeptídeos/metabolismo , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/tratamento farmacológico , Internalização do Vírus/efeitos dos fármacos
14.
PLoS Biol ; 17(7): e3000367, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31323018

RESUMO

Human papillomaviruses (HPVs) are causative agents of various diseases associated with cellular hyperproliferation, including cervical cancer, one of the most prevalent tumors in women. E7 is one of the two HPV-encoded oncoproteins and directs recruitment and subsequent degradation of tumor-suppressive proteins such as retinoblastoma protein (pRb) via its LxCxE motif. E7 also triggers tumorigenesis in a pRb-independent pathway through its C-terminal domain, which has yet been largely undetermined, with a lack of structural information in a complex form with a host protein. Herein, we present the crystal structure of the E7 C-terminal domain of HPV18 belonging to the high-risk HPV genotypes bound to the catalytic domain of human nonreceptor-type protein tyrosine phosphatase 14 (PTPN14). They interact directly and potently with each other, with a dissociation constant of 18.2 nM. Ensuing structural analysis revealed the molecular basis of the PTPN14-binding specificity of E7 over other protein tyrosine phosphatases and also led to the identification of PTPN21 as a direct interacting partner of E7. Disruption of HPV18 E7 binding to PTPN14 by structure-based mutagenesis impaired E7's ability to promote keratinocyte proliferation and migration. Likewise, E7 binding-defective PTPN14 was resistant for degradation via proteasome, and it was much more effective than wild-type PTPN14 in attenuating the activity of downstream effectors of Hippo signaling and negatively regulating cell proliferation, migration, and invasion when examined in HPV18-positive HeLa cells. These results therefore demonstrated the significance and therapeutic potential of the intermolecular interaction between HPV E7 and host PTPN14 in HPV-mediated cell transformation and tumorigenesis.


Assuntos
Transformação Celular Neoplásica , Proteínas de Ligação a DNA/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Neoplasias do Colo do Útero/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Feminino , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/genética , Ligação Proteica , Domínios Proteicos , Proteínas Tirosina Fosfatases não Receptoras/química , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteína do Retinoblastoma/química , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Homologia de Sequência de Aminoácidos , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
15.
Virus Genes ; 55(5): 600-609, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31290065

RESUMO

Human papillomaviruses (HPVs) of genus betapapillomavirus (betaHPV) are implicated in skin carcinogenesis, but their exact role in keratinocyte transformation is poorly understood. We show an interaction of HPV5 and HPV8 oncoproteins E6 and E7 with the nuclear mitotic apparatus protein 1 (NuMA). Binding of E6 or E7 to NuMA induces little aneuploidy, cell cycle alterations, or aberrant centrosomes. Intracellular localization of NuMA is not altered by E6 and E7 expression in 2D cultures. However, the localization profile is predominantly cytoplasmic in 3D organotypic skin models. Both viral proteins colocalize with NuMA in interphase cells, while only E7 colocalizes with NuMA in mitotic cells. Intriguingly, a small subset of cells shows E7 at only one spindle pole, whereas NuMA is present at both poles. This dissimilar distribution of E7 at the spindle poles may alter cell differentiation, which may in turn be relevant for betaHPV-induced skin carcinogenesis.


Assuntos
Betapapillomavirus/crescimento & desenvolvimento , Proteínas de Ciclo Celular/metabolismo , Interações Hospedeiro-Patógeno , Queratinócitos/virologia , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Humanos , Ligação Proteica , Mapeamento de Interação de Proteínas
16.
Chem Commun (Camb) ; 55(61): 8959-8962, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31290487

RESUMO

Hydrocarbon stapled peptides are promising therapeutics for inhibition of intracellular protein-protein interactions. Here we develop a new high-throughput strategy for hydrocarbon stapled peptide discovery based on mRNA display of peptides containing α-methyl cysteine and cyclized with m-dibromoxylene. We focus on development of a peptide binder to the HPV16 E2 protein.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Evolução Molecular Direcionada/métodos , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Fatores de Transcrição/metabolismo , Alquilação , Sequência de Aminoácidos , Proteínas de Ciclo Celular , Ciclização , Cisteína/química , Hidrocarbonetos Bromados/química , Biblioteca de Peptídeos , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/química
17.
Curr Protein Pept Sci ; 20(9): 926-934, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31244421

RESUMO

Human papillomavirus (HPV) cancers are expected to be major global health concerns in the upcoming decades. The growth of HPV-positive cancer cells depends on the consistent expression of oncoprotein which has been poorly taken into account in the cellular communication. Among them, E6/E7 oncoproteins are attractive therapeutic targets as their inhibition rapidly leads to the onset of aging in HPV-positive cancer cells. This cellular response is associated with the regeneration of p53, pRb anti-proliferative proteins as well as the mTOR signaling pathway; hence, the identification of involved and application of E6/E7 inhibitors can lead to new therapeutic strategies. In the present review, we focused on the pathogenicity of E6/E7 Proteins of human papillomavirus and their roles associated with the cervical cancer.


Assuntos
Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/virologia , Neoplasias do Colo do Útero/etiologia , Ciclo Celular , Metabolismo Energético , Feminino , Humanos , Imunidade Inata , Proteínas Oncogênicas Virais/imunologia , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/imunologia , Proteínas E7 de Papillomavirus/imunologia , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/imunologia , Transdução de Sinais , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia
18.
Vet Microbiol ; 233: 39-46, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176410

RESUMO

E5 protein, the major oncoprotein of bovine Deltapapillomavirus (BPV), was found to be expressed in 18 of 21 examined urothelial cancers of cattle. E5 oncoprotein was found to interact with p62 which was degraded through the autophagosome-lysosome pathway as well as LC3-II and appeared to be involved in the phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α). Autophagy was morphologically documented by transmission electron microscope (TEM) through the detection of double-membrane autophagosomes and autolysosomes. Overexpression of Bag3 known to mediate selective autophagy was also demonstrated. Furthermore, Bag3 and BPV E5 oncoprotein were seen to co-localize with dynein and 14-3-3γ, which suggested that Bag3 could be involved in inducing the retrograde transport of BPV E5 along microtubules to aggresomes, perinuclear sites with high autophagic flux. Electron dense perinuclear structures consistent with aggresomes were also documented by TEM in urothelial cancer cells. Finally, Bag3 was found to also interact with synaptopodin 2 (Synpo2), which would seem to contribute to cargo degradation as it has been shown to facilitate autophagosome formation. This study provides mechanistic insights into the potential role(s) of autophagy in BPV disease, which can help to develop future treatment and control measures for BPV infection. Activation of autophagy correlates positively with BPV infection and may play a role in biological behavior of bladder cancer as urothelial carcinomas of cattle are known to be characterized by a relatively low rate of metastasis.


Assuntos
Autofagia , Papillomavirus Bovino 1/genética , Expressão Gênica , Proteínas Oncogênicas Virais/genética , Neoplasias da Bexiga Urinária/veterinária , Animais , Bovinos , Doenças dos Bovinos/virologia , DNA Viral/genética , DNA Viral/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Redes Reguladoras de Genes , Interações entre Hospedeiro e Microrganismos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Fosforilação , Neoplasias da Bexiga Urinária/virologia , Urotélio/virologia
19.
Int J Mol Sci ; 20(9)2019 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-31058807

RESUMO

Human papillomavirus (HPV)-induced cancers will remain a significant clinical challenge for decades. Thus, the development of novel treatment strategies is urgently required, which should benefit from improving our understanding of the mechanisms of HPV-induced cell transformation. This should also include analyses of hypoxic tumor cells, which represent a major problem for cancer therapy. Recent evidence indicates that the PI3K/AKT/mTOR network plays a key role for the virus/host cell crosstalk in both normoxic and hypoxic HPV-positive cancer cells. In normoxic cells, the efficacy of the senescence induction upon experimental E6/E7 repression depends on active mTORC1 signaling. Under hypoxia, however, HPV-positive cancer cells can evade senescence due to hypoxic impairment of mTORC1 signaling, albeit the cells strongly downregulate E6/E7. Hypoxic repression of E6/E7 is mediated by the AKT kinase, which is activated under hypoxia by its canonical upstream regulators mTORC2 and PI3K. This review highlights our current knowledge about the oxygen-dependent crosstalk of the PI3K/AKT/mTOR signaling circuit with the HPV oncogenes and the phenotypic state of the host cell. Moreover, since the PI3K/AKT/mTOR pathway is considered to be a promising target for anticancer therapy, we discuss clinical implications for the treatment of HPV-positive cervical and head and neck squamous cell carcinomas.


Assuntos
Interações Hospedeiro-Patógeno , Infecções por Papillomavirus/complicações , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Neoplasias do Colo do Útero/etiologia , Neoplasias do Colo do Útero/metabolismo , Alphapapillomavirus/fisiologia , Animais , Suscetibilidade a Doenças , Feminino , Humanos , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/virologia
20.
PLoS Pathog ; 15(5): e1007769, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31116803

RESUMO

The Human Papillomavirus E7 oncoprotein plays an essential role in the development and maintenance of malignancy, which it achieves through targeting a number of critical cell control pathways. An important element in the ability of E7 to contribute towards cell transformation is the presence of a Casein Kinase II phospho-acceptor site within the CR2 domain of the protein. Phosphorylation is believed to enhance E7 interaction with a number of different cellular target proteins, and thereby increase the ability of E7 to enhance cell proliferation and induce malignancy. However, there is little information on how important this site in E7 is, once the tumour cells have become fully transformed. In this study, we have performed genome editing of the HPV-18 E7 CKII recognition site in C4-1 cervical tumour-derived cells. We first show that mutation of HPV18 E7 S32/S34 to A32/A34 abolishes CKII phosphorylation of E7, and subsequently we have isolated C4-1 clones containing these mutations in E7. The cells continue to proliferate, but are somewhat more slow-growing than wild type cells, reach lower saturation densities, and are also more susceptible to low nutrient conditions. These cells are severely defective in matrigel invasion assays, partly due to downregulation of matrix metalloproteases (MMPs). Mechanistically, we find that phosphorylation of E7 plays a direct role in the ability of E7 to activate AKT signaling, which in turn is required for optimal levels of MMP secretion. These results demonstrate that the E7 CKII phospho-acceptor site thus continues to play an important role for E7's activity in cells derived from cervical cancers, and suggests that blocking this activity of E7 could be expected to have therapeutic potential.


Assuntos
Caseína Quinase II/metabolismo , Proliferação de Células , Transformação Celular Viral , Proteínas de Ligação a DNA/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Neoplasias do Colo do Útero/patologia , Caseína Quinase II/genética , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Proteínas Oncogênicas Virais/genética , Fenótipo , Fosforilação , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo
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