RESUMEN
Merkel Cell Carcinoma (MCC) is a rare neuroendocrine skin cancer. In our previous work, we decoded genes specifically deregulated by MCPyV early genes as opposed to other polyomaviruses and established functional importance of NDRG1 in inhibiting cellular proliferation and migration in MCC. In the present work, we found the SET protein, (I2PP2A, intrinsic inhibitor of PP2A) upstream of NDRG1 which was modulated by MCPyV early genes, both in hTERT-HK-MCPyV and MCPyV-positive (+) MCC cell lines. Additionally, MCC dermal tumour nodule tissues showed strong SET expression. Inhibition of the SET-PP2A interaction in hTERT-HK-MCPyV using the small molecule inhibitor, FTY720, increased NDRG1 expression and inhibited cell cycle regulators, cyclinD1 and CDK2. SET inhibition by shRNA and FTY720 also decreased cell proliferation and colony formation in MCPyV(+) MCC cells. Overall, these results pave a path for use of drugs targeting SET protein for the treatment of MCC.
Asunto(s)
Carcinoma de Células de Merkel , Movimiento Celular , Proliferación Celular , Poliomavirus de Células de Merkel , Proteína Fosfatasa 2 , Humanos , Poliomavirus de Células de Merkel/fisiología , Poliomavirus de Células de Merkel/genética , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/genética , Carcinoma de Células de Merkel/virología , Carcinoma de Células de Merkel/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Clorhidrato de Fingolimod/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Línea Celular Tumoral , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/genética , Infecciones por Polyomavirus/virología , Neoplasias Cutáneas/virología , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 2 Dependiente de la Ciclina/genéticaRESUMEN
Epstein-Barr Virus (EBV) infects more than 90% of the adult population worldwide. EBV infection is associated with Burkitt lymphoma (BL) though alone is not sufficient to induce carcinogenesis implying the involvement of co-factors. BL is endemic in African regions faced with mycotoxins exposure. Exposure to mycotoxins and oncogenic viruses has been shown to increase cancer risks partly through the deregulation of the immune response. A recent transcriptome profiling of B cells exposed to aflatoxin B1 (AFB1) revealed an upregulation of the Chemokine ligand 22 (CCL22) expression although the underlying mechanisms were not investigated. Here, we tested whether mycotoxins and EBV exposure may together contribute to endemic BL (eBL) carcinogenesis via immunomodulatory mechanisms involving CCL22. Our results revealed that B cells exposure to AFB1 and EBV synergistically stimulated CCL22 secretion via the activation of Nuclear Factor-kappa B pathway. By expressing EBV latent genes in B cells, we revealed that elevated levels of CCL22 result not only from the expression of the latent membrane protein LMP1 as previously reported but also from the expression of other viral latent genes. Importantly, CCL22 overexpression resulting from AFB1-exposure in vitro increased EBV infection through the activation of phosphoinositide-3-kinase pathway. Moreover, inhibiting CCL22 in vitro and in humanized mice in vivo limited EBV infection and decreased viral genes expression, supporting the notion that CCL22 overexpression plays an important role in B cell infection. These findings unravel new mechanisms that may underpin eBL development and identify novel pathways that can be targeted in drug development.
Asunto(s)
Linfoma de Burkitt , Infecciones por Virus de Epstein-Barr , Animales , Ratones , Herpesvirus Humano 4/genética , Infecciones por Virus de Epstein-Barr/complicaciones , Aflatoxina B1/toxicidad , Ligandos , Linfoma de Burkitt/metabolismo , Quimiocinas , CarcinogénesisRESUMEN
Burkitt lymphoma (BL) is a malignant B cell neoplasm that accounts for almost half of pediatric cancers in sub-Saharan African countries. Although the BL endemic prevalence is attributable to the combination of Epstein-Barr virus (EBV) infection with malaria and environmental carcinogens exposure, such as the food contaminant aflatoxin B1 (AFB1), the molecular determinants underlying the pathogenesis are not fully understood. Consistent with the role of epigenetic mechanisms at the interface between the genome and environment, AFB1 and EBV impact the methylome of respectively leukocytes and B cells specifically. Here, we conducted a thorough investigation of common epigenomic changes following EBV or AFB1 exposure in B cells. Genome-wide DNA methylation profiling identified an EBV-AFB1 common signature within the TGFBI locus, which encodes for a putative tumor suppressor often altered in cancer. Subsequent mechanistic analyses confirmed a DNA-methylation-dependent transcriptional silencing of TGFBI involving the recruitment of DNMT1 methyltransferase that is associated with an activation of the NF-κB pathway. Our results reveal a potential common mechanism of B cell transformation shared by the main risk factors of endemic BL (EBV and AFB1), suggesting a key determinant of disease that could allow the development of more efficient targeted therapeutic strategies.
RESUMEN
Tumor suppressors can exert pro-proliferation functions in specific contexts. In the beta human papillomavirus type 38 (HPV38) experimental model, the viral proteins E6 and E7 promote accumulation of a wild-type (WT) p53 form in human keratinocytes (HKs), promoting cellular proliferation. Inactivation of p53 by different means strongly decreases the proliferation of HPV38 E6/E7 HKs. This p53 form is phosphorylated at S392 by the double-stranded RNA-dependent protein kinase PKR, which is highly activated by HPV38. PKR-mediated S392 p53 phosphorylation promotes the formation of a p53/DNMT1 complex, which inhibits expression of integrin alpha 1 (ITGA1), a repressor of epidermal growth factor receptor (EGFR) signaling. Ectopic expression of ITGA1 in HPV38 E6/E7 HKs promotes EGFR degradation, inhibition of cellular proliferation, and cellular death. Itga1 expression was also inhibited in the skin of HPV38 transgenic mice that have an elevated susceptibility to UV-induced skin carcinogenesis. In summary, these findings reveal the existence of a specific WT p53 form that displays pro-proliferation properties.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Proliferación Celular , Queratinocitos/patología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Infecciones por Papillomavirus/complicaciones , Proteínas Represoras/metabolismo , Neoplasias Cutáneas/etiología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Células Cultivadas , Regulación hacia Abajo , Humanos , Queratinocitos/inmunología , Queratinocitos/virología , Ratones , Ratones Transgénicos , Proteínas Oncogénicas Virales/genética , Papillomaviridae/aislamiento & purificación , Proteínas E7 de Papillomavirus/genética , Infecciones por Papillomavirus/virología , Proteínas Represoras/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The beta human papillomaviruses (HPVs) are subdivided into 5 species (beta-1 to beta-5), and they were first identified in the skin. However, the beta-3 species appears to be more highly represented in the mucosal epithelia than in the skin. Functional studies have also highlighted that beta-3 HPV49 shares some functional similarities with mucosal high-risk (HR) HPV16. Here, we describe the characterization of the in vitro transforming properties of the entire beta-3 species, which includes three additional HPV types: HPV75, HPV76, and HPV115. HPV49, HPV75, and HPV76 E6 and E7 (E6/E7), but not HPV115 E6 and E7, efficiently inactivate the p53 and pRb pathways and immortalize or extend the life span of human foreskin keratinocytes (HFKs). As observed for HR HPV16, cell cycle deregulation mediated by beta-3 HPV E6/E7 expression leads to p16INK4a accumulation, whereas no p16INK4a was detected in beta-2 HPV38 E6/E7 HFKs. As shown for HPV49 E6, HPV75 and HPV76 E6s degrade p53 by an E6AP/proteasome-mediated mechanism. Comparative analysis of cellular gene expression patterns of HFKs containing E6 and E7 from HR HPV16, beta-3 HPV types, and beta-2 HPV38 further highlights the functional similarities of HR HPV16 and beta-3 HPV49, HPV75, and HPV76. The expression profiles of these four HPV HFKs show some similarities and diverge substantially from those of beta-3 HPV115 E6/E7 and beta-2 HPV38 E6/E7 HFKs. In summary, our data show that beta-3 HPV types share some mechanisms with HR HPV types and pave the way for additional studies aiming to evaluate their potential role in human pathologies.IMPORTANCE Human papillomaviruses are currently classified in different genera. Mucosal HPVs belonging to the alpha genus have been clearly associated with carcinogenesis of the mucosal epithelium at different sites. Beta HPV types have been classified as cutaneous. Although findings indicate that some beta HPVs from species 1 and 2 play a role, together with UV irradiation, in skin cancer, very little is known about the transforming properties of most of the beta HPVs. This report shows the transforming activity of E6 and E7 from beta-3 HPV types. Moreover, it highlights that beta-3 HPVs share some biological properties more extensively with mucosal high-risk HPV16 than with beta-2 HPV38. This report provides new paradigms for a better understanding of the biology of the different HPV types and their possible association with lesions at mucosal and/or cutaneous epithelia.
Asunto(s)
Alphapapillomavirus/genética , Alphapapillomavirus/patogenicidad , Células Epiteliales/virología , Membrana Mucosa/virología , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/genética , Alphapapillomavirus/clasificación , Animales , Células Cultivadas , Papillomavirus Humano 16/genética , Humanos , Queratinocitos/virología , Masculino , Ratones , Membrana Mucosa/citología , Células 3T3 NIH , Piel/virologíaRESUMEN
Merkel cell polyomavirus (MCPyV) is the first human polyomavirus etiologically associated with Merkel cell carcinoma (MCC), a rare and aggressive form of skin cancer. Similar to other polyomaviruses, MCPyV encodes early T antigen genes, viral oncogenes required for MCC tumor growth. To identify the unique oncogenic properties of MCPyV, we analyzed the gene expression profiles in human spontaneously immortalized keratinocytes (NIKs) expressing the early genes from six distinct human polyomaviruses (PyVs), including MCPyV. A comparison of the gene expression profiles revealed 28 genes specifically deregulated by MCPyV. In particular, the MCPyV early gene downregulated the expression of the tumor suppressor gene N-myc downstream-regulated gene 1 (NDRG1) in MCPyV gene-expressing NIKs and hTERT-MCPyV gene-expressing human keratinocytes (HK) compared to their expression in the controls. In MCPyV-positive MCC cells, the expression of NDRG1 was downregulated by the MCPyV early gene, as T antigen knockdown rescued the level of NDRG1. In addition, NDRG1 overexpression in hTERT-MCPyV gene-expressing HK or MCC cells resulted in a decrease in the number of cells in S phase and cell proliferation inhibition. Moreover, a decrease in wound healing capacity in hTERT-MCPyV gene-expressing HK was observed. Further analysis revealed that NDRG1 exerts its biological effect in Merkel cell lines by regulating the expression of the cyclin-dependent kinase 2 (CDK2) and cyclin D1 proteins. Overall, NDRG1 plays an important role in MCPyV-induced cellular proliferation.IMPORTANCE Merkel cell carcinoma was first described in 1972 as a neuroendocrine tumor of skin, most cases of which were reported in 2008 to be caused by a PyV named Merkel cell polyomavirus (MCPyV), the first PyV linked to human cancer. Thereafter, numerous studies have been conducted to understand the etiology of this virus-induced carcinogenesis. However, it is still a new field, and much work is needed to understand the molecular pathogenesis of MCC. In the current work, we sought to identify the host genes specifically deregulated by MCPyV, as opposed to other PyVs, in order to better understand the relevance of the genes analyzed on the biological impact and progression of the disease. These findings open newer avenues for targeted drug therapies, thereby providing hope for the management of patients suffering from this highly aggressive cancer.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Movimiento Celular/genética , Proliferación Celular/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Poliomavirus de Células de Merkel/genética , Poliomavirus de Células de Merkel/fisiología , Antígenos Virales de Tumores/genética , Antígenos Virales de Tumores/metabolismo , Carcinogénesis/genética , Carcinoma de Células de Merkel/virología , Línea Celular , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica , Humanos , Queratinocitos/virología , Infecciones por Polyomavirus/virología , Piel/patología , Neoplasias Cutáneas/genética , Transcriptoma , Infecciones Tumorales por Virus/virologíaRESUMEN
The histone modifier lysine (K)-specific demethylase 2B (KDM2B) plays a role in the differentiation of hematopoietic cells, and its expression appears to be deregulated in certain cancers of hematological and lymphoid origins. We have previously found that the KDM2B gene is differentially methylated in cell lines derived from Epstein-Barr virus (EBV)-associated endemic Burkitt lymphoma (eBL) compared with that in EBV-negative sporadic Burkitt lymphoma-derived cells. However, whether KDM2B plays a role in eBL development has not been previously investigated. Oncogenic viruses have been shown to hijack the host cell epigenome to complete their life cycle and to promote the transformation process by perturbing cell chromatin organization. Here, we investigated whether EBV alters KDM2B levels to enable its life cycle and promote B-cell transformation. We show that infection of B cells with EBV leads to downregulation of KDM2B levels. We also show that LMP1, one of the main EBV transforming proteins, induces increased DNMT1 recruitment to the KDM2B gene and augments its methylation. By altering KDM2B levels and performing chromatin immunoprecipitation in EBV-infected B cells, we show that KDM2B is recruited to the EBV gene promoters and inhibits their expression. Furthermore, forced KDM2B expression in immortalized B cells led to altered mRNA levels of some differentiation-related genes. Our data show that EBV deregulates KDM2B levels through an epigenetic mechanism and provide evidence for a role of KDM2B in regulating virus and host cell gene expression, warranting further investigations to assess the role of KDM2B in the process of EBV-mediated lymphomagenesis.IMPORTANCE In Africa, Epstein-Barr virus infection is associated with endemic Burkitt lymphoma, a pediatric cancer. The molecular events leading to its development are poorly understood compared with those leading to sporadic Burkitt lymphoma. In a previous study, by analyzing the DNA methylation changes in endemic compared with sporadic Burkitt lymphoma cell lines, we identified several differential methylated genomic positions in the proximity of genes with a potential role in cancer, and among them was the KDM2B gene. KDM2B encodes a histone H3 demethylase already shown to be involved in some hematological disorders. However, whether KDM2B plays a role in the development of Epstein-Barr virus-mediated lymphoma has not been investigated before. In this study, we show that Epstein-Barr virus deregulates KDM2B expression and describe the underlying mechanisms. We also reveal a role of the demethylase in controlling viral and B-cell gene expression, thus highlighting a novel interaction between the virus and the cellular epigenome.
Asunto(s)
Epigénesis Genética , Infecciones por Virus de Epstein-Barr/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Herpesvirus Humano 4/fisiología , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Adolescente , Adulto , Linfocitos B/virología , Linfoma de Burkitt/metabolismo , Línea Celular , Niño , Preescolar , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Metilación de ADN , Regulación hacia Abajo , Infecciones por Virus de Epstein-Barr/genética , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
An increasing number of studies suggest that cutaneous papillomaviruses (PVs) might be involved in skin carcinogenesis. However, only a few animal PVs have been investigated regard to their transformation properties. Here, we investigate and compare the oncogenic potential of 2 ovine Delta and Dyokappa PVs, isolated from ovine skin lesions, in vitro and ex vivo. We demonstrate that both OaPV4 (Delta) and OaPV3 (Dyokappa) E6 and E7 immortalize primary sheep keratinocytes and efficiently deregulate pRb pathway, although they seem unable to alter p53 activity. Moreover, OaPV3 and OaPV4-E6E7 expressing cells show different shape, doubling time, and clonogenic activities, providing evidence for a stronger transforming potential of OaPV3 respect to OaPV4. Also, similarly to high-risk mucosal and cutaneous PVs, the OaPV3-E7 protein, constantly expressed in sheep squamous cell carcinomas, binds pRb with higher affinity compared to the E7 encoded by OaPV4, a virus associated to fibropapilloma. Finally, we found that OaPV3 and OaPV4-E6E7 determine upregulation of the pro-proliferative proteins cyclin A and cdk1 in both human and ovine primary keratinocytes. Collectively, results provide evidence for implication of ovine PVs in cutaneous proliferative lesions and skin cancer progression, and indicate sheep as a possible animal model for the study of cutaneous lesions and malignancies.
Asunto(s)
Queratinocitos/virología , Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Proteínas E7 de Papillomavirus/genética , Piel/virología , Transformación Genética , Animales , Proteína Quinasa CDC2/genética , Células Cultivadas , Ciclina A/genética , Deltapapillomavirus/genética , Deltapapillomavirus/aislamiento & purificación , Humanos , Ratones , Células 3T3 NIH , Ovinos , Piel/patología , Regulación hacia ArribaRESUMEN
Cutaneous beta human papillomavirus (HPV) types are suspected to be involved, together with ultraviolet (UV) radiation, in the development of non-melanoma skin cancer (NMSC). Studies in in vitro and in vivo experimental models have highlighted the transforming properties of beta HPV E6 and E7 oncoproteins. However, epidemiological findings indicate that beta HPV types may be required only at an initial stage of carcinogenesis, and may become dispensable after full establishment of NMSC. Here, we further investigate the potential role of beta HPVs in NMSC using a Cre-loxP-based transgenic (Tg) mouse model that expresses beta HPV38 E6 and E7 oncogenes in the basal layer of the skin epidermis and is highly susceptible to UV-induced carcinogenesis. Using whole-exome sequencing, we show that, in contrast to WT animals, when exposed to chronic UV irradiation K14 HPV38 E6/E7 Tg mice accumulate a large number of UV-induced DNA mutations, which increase proportionally with the severity of the skin lesions. The mutation pattern detected in the Tg skin lesions closely resembles that detected in human NMSC, with the highest mutation rate in p53 and Notch genes. Using the Cre-lox recombination system, we observed that deletion of the viral oncogenes after development of UV-induced skin lesions did not affect the tumour growth. Together, these findings support the concept that beta HPV types act only at an initial stage of carcinogenesis, by potentiating the deleterious effects of UV radiation.
Asunto(s)
Carcinogénesis/efectos de la radiación , Neoplasias Inducidas por Radiación/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Neoplasias Cutáneas/metabolismo , Piel/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Proteínas Virales/metabolismo , Animales , Betapapillomavirus/metabolismo , Epidermis/metabolismo , Epidermis/patología , Epidermis/efectos de la radiación , Femenino , Eliminación de Gen , Genes p53/efectos de la radiación , Ratones , Ratones Transgénicos , Mutagénesis/efectos de la radiación , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Inducidas por Radiación/patología , Proteínas Oncogénicas Virales/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Recombinantes/metabolismo , Piel/metabolismo , Piel/patología , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/patología , Carga Tumoral/efectos de la radiación , Proteínas Virales/genéticaRESUMEN
The connection between chronic inflammation and risk of cancer has been supported by several studies. The development of cancer might be a process driven by the presence of a specific combination of inflammatory mediators, including cytokines, chemokines and enzymes, in the tumor microenvironment. Virus-induced tumors, like HPV-induced Squamous Cell Carcinomas, represent a paradigmatic example of the interplay between inflammation, as integral part of the innate antiviral response, and malignant transformation. Here, the role of inflammatory microenvironment in the HPV-induced carcinogenesis is addressed, with a specific focus on the involvement of the immune molecules as well as their delivery through the microvesicle cargo possibly correlated to the different HPV genotype. The expression of the inflammatory mediators in HPV positive cells has been analyzed in primary human foreskin keratinocytes and keratinocytes transduced by E6 and E7 from mucosal HPV-16 or cutaneous HPV-38 genotypes. HPV E6 and E7 proteins can modulate the expression of immune mediators in HPV-infected cells and can affect the levels of immune molecules, mainly chemokines, in the extracellular milieu. HPV-16 E6 and E7 oncoproteins have been silenced to confirm the specificity of the modulation of the inflammatory microenvironment. Our results suggest that the expression of HPV oncoproteins allows the modification of the tumor milieu through the synthesis and release of specific pro-inflammatory cytokines and chemokines, affecting the efficacy of the immune response. The microenvironment can also be conditioned by an altered mRNA cargo delivered by extracellular vesicles, thereby efficiently affecting the surrounding cells with possible implication for tumorigenesis and tumor diagnosis.
Asunto(s)
Microambiente Celular , Vesículas Extracelulares/metabolismo , Mediadores de Inflamación/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Represoras/metabolismo , Línea Celular , Silenciador del Gen , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Several lines of evidence indicate that cutaneous human papillomavirus (HPV) types belonging to the beta genus of the HPV phylogenetic tree synergize with UV radiation in the development of skin cancer. Accordingly, the E6 and E7 oncoproteins from some beta HPV types are able to deregulate pathways related to immune response and cellular transformation. Toll-like receptor 9 (TLR9), in addition to playing a role in innate immunity, has been shown to be involved in the cellular stress response. Using primary human keratinocytes as experimental models, we have shown that UV irradiation (and other cellular stresses) activates TLR9 expression. This event is closely linked to p53 activation. Silencing the expression of p53 or deleting its encoding gene affected the activation of TLR9 expression after UV irradiation. Using various strategies, we have also shown that the transcription factors p53 and c-Jun are recruited onto a specific region of the TLR9 promoter after UV irradiation. Importantly, the E6 and E7 oncoproteins from beta HPV38, by inducing the accumulation of the p53 antagonist ΔNp73α, prevent the UV-mediated recruitment of these transcription factors onto the TLR9 promoter, with subsequent impairment of TLR9 gene expression. This study provides new insight into the mechanism that mediates TLR9 upregulation in response to cellular stresses. In addition, we show that HPV38 E6 and E7 are able to interfere with this mechanism, providing another explanation for the possible cooperation of beta HPV types with UV radiation in skin carcinogenesis.IMPORTANCE Beta HPV types have been suggested to act as cofactors in UV-induced skin carcinogenesis by altering several cellular mechanisms activated by UV radiation. We show that the expression of TLR9, a sensor of damage-associated molecular patterns produced during cellular stress, is activated by UV radiation in primary human keratinocytes (PHKs). Two transcription factors known to be activated by UV radiation, p53 and c-Jun, play key roles in UV-activated TLR9 expression. The E6 and E7 oncoproteins from beta HPV38 strongly inhibit UV-activated TLR9 expression by preventing the recruitment of p53 and c-Jun to the TLR9 promoter. Our findings provide additional support for the role that beta HPV types play in skin carcinogenesis by preventing activation of specific pathways upon exposure of PHKs to UV radiation.
Asunto(s)
Transformación Celular Neoplásica/patología , Activación Enzimática/efectos de la radiación , Queratinocitos/metabolismo , Papillomaviridae/crecimiento & desarrollo , Proteínas E7 de Papillomavirus/metabolismo , Receptor Toll-Like 9/metabolismo , Receptor Toll-Like 9/efectos de la radiación , Proteínas Virales/metabolismo , Proliferación Celular/genética , Células Cultivadas , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Regiones Promotoras Genéticas/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Piel/parasitología , Piel/virología , Neoplasias Cutáneas/virología , Receptor Toll-Like 9/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Rayos UltravioletaRESUMEN
Epstein-Barr virus (EBV) was identified as the first human virus to be associated with a human malignancy, Burkitt's lymphoma (BL), a pediatric cancer endemic in sub-Saharan Africa. The exact mechanism of how EBV contributes to the process of lymphomagenesis is not fully understood. Recent studies have highlighted a genetic difference between endemic (EBV+) and sporadic (EBV-) BL, with the endemic variant showing a lower somatic mutation load, which suggests the involvement of an alternative virally-driven process of transformation in the pathogenesis of endemic BL. We tested the hypothesis that a global change in DNA methylation may be induced by infection with EBV, possibly thereby accounting for the lower mutation load observed in endemic BL. Our comparative analysis of the methylation profiles of a panel of BL derived cell lines, naturally infected or not with EBV, revealed that the presence of the virus is associated with a specific pattern of DNA methylation resulting in altered expression of cellular genes with a known or potential role in lymphomagenesis. These included ID3, a gene often found to be mutated in sporadic BL. In summary this study provides evidence that EBV may contribute to the pathogenesis of BL through an epigenetic mechanism.
Asunto(s)
Linfoma de Burkitt/genética , Linfoma de Burkitt/virología , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Herpesvirus Humano 4/fisiología , Linfoma de Burkitt/patología , Línea Celular Tumoral , Islas de CpG/genética , Metilación de ADN/genética , Regulación hacia Abajo/genética , Silenciador del Gen , Humanos , Proteínas Inhibidoras de la Diferenciación/metabolismo , Mutación/genética , Proteínas de Neoplasias/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de la Matriz Viral/metabolismoRESUMEN
BACKGROUND: Head and neck squamous cell carcinomas (HNSCCs) represent a heterogeneous group of cancers for which human papilloma virus (HPV) infection is an emerging risk factor. Previous studies showed promoter hypermethylation in HPV(+) oropharyngeal cancers, but only few consistent target genes have been so far described, and the evidence of a functional impact on gene expression is still limited. METHODS: We performed global and stratified pooled analyses of epigenome-wide data in HNSCCs based on the Illumina HumanMethylation450 bead-array data in order to identify tissue-specific components and common viral epigenetic targets in HPV-associated tumours. RESULTS: We identified novel differentially methylated CpGs and regions associated with viral infection that are independent of the anatomic site. In particular, most hypomethylated regions were characterized by a marked loss of CpG island boundaries, which showed significant correlations with expression of neighbouring genes. Moreover, a subset of only five CpGs in a few hypomethylated regions predicted HPV status with a high level of specificity in different cohorts. Finally, this signature was a better predictor of survival compared with HPV status determined by viral gene expression by RNA sequencing in The Cancer Genome Atlas cohort. CONCLUSIONS: We identified a novel epigenetic signature of HPV infection in HNSCCs which is independent of the anatomic site, is functionally correlated with gene expression and may be leveraged for improved stratification of prognosis in HNSCCs.
Asunto(s)
Carcinoma de Células Escamosas/genética , Metilación de ADN , Neoplasias de Cabeza y Cuello/genética , Infecciones por Papillomavirus/complicaciones , Regiones Promotoras Genéticas , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/etiología , Carcinoma de Células Escamosas/metabolismo , Islas de CpG , Femenino , Regulación de la Expresión Génica , Genoma Humano , Neoplasias de Cabeza y Cuello/etiología , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Carcinoma de Células Escamosas de Cabeza y Cuello , Adulto JovenRESUMEN
Human Papilloma Viruses (HPVs) are the causative agents of cervical cancer although other types of cancers are associated with HPV infection. Type I Interferons can interfere with HPV E6- and/or E7-dependent transformation and can affect microRNA (miRNA) expression. Cancer cells show a specific pattern of miRNA expression and HPVs are able to modulate miRNAs expressed in infected cells. Keratinocytes transduced with E6 and E7 from mucosal HPV-16 or cutaneous HPV-38 (K16 and K38) were studied to analyze the involvement of HPV oncoproteins in the anti-proliferative activity of IFN-ß. In view of our previous data showing senescence induction by the cytokine in K38 cells, we observe that IFN-ß treatment leads to p53-indipendent apoptosis in K16 cells whereas induces senescence in K16 cells if E6 is silenced and p53 expression is restored. The levels of selected miRNAs, deregulated in K16 and K38 cells, can be modulated by IFN-ß when E6 and E7 proteins of HPV-16, but not HPV-38, are expressed.
Asunto(s)
Apoptosis/efectos de los fármacos , Papillomavirus Humano 16/metabolismo , Interferón beta/farmacología , Queratinocitos/metabolismo , MicroARNs/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Represoras/metabolismo , Apoptosis/genética , Línea Celular Transformada , Papillomavirus Humano 16/genética , Humanos , Queratinocitos/patología , Queratinocitos/virología , MicroARNs/genética , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/genética , Proteínas Represoras/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
PURPOSE: Human papillomaviruses (HPVs) are the causative agents of cervical cancer and are also associated with other types of cancers. HPVs can modulate microRNAs (miRNAs) expressed by infected cells. The production of extracellular vesicles is deregulated in cancer, and their cargo delivered to the microenvironment can promote tumorigenesis. The involvement of HPV oncoproteins on miRNA expression in cells and exosomes was analyzed in keratinocytes transduced with E6 and E7 from mucosal HPV-16 or cutaneous HPV-38 (K16 and K38). METHODS: MiRNAs were investigated through the TaqMan Array Human MicroRNA Cards, followed by real-time RT-PCR assay for specific miRNAs. Selected miRNA targets were analyzed by Western blot and correlated to the HPV oncoproteins by specifically silencing E6 and E7 expression. Exosomes, isolated from K16 and K38 supernatants by differential centrifugations, were quantified through the vesicle-associated acetylcholinesterase activity. RESULTS: MiRNAs deregulated in K16 and K38 cells were identified. HPV-16 and/or HPV-38 E6 and E7 single proteins can modify the expression of selected miRNAs involved in the tumorigenesis, in particular miR-18a, -19a, -34a and -590-5p. The analysis of the content of exosomes isolated from HPV-positive cells revealed the presence of E6 and E7 mRNAs and few miRNAs. MiR-222, a key miRNA deregulated in many cancers, was identified in exosomes from K16 cells. CONCLUSIONS: HPV E6 and/or E7 oncoprotein expression can induce the deregulation of some miRNAs. Through the production and function of exosomes, HPV oncogenes as well as HPV-deregulated miRNAs can potentiate the virus oncogenic effects in the tumor cell microenvironment.
Asunto(s)
Carcinogénesis , MicroARNs/genética , Neoplasias/genética , Proteínas Oncogénicas Virales/metabolismo , Humanos , Neoplasias/virologíaRESUMEN
Felis catus papillomavirus type 2 (FcaPV2) DNA is found in feline cutaneous squamous cell carcinomas (SCCs); however, its biological properties are still uncharacterized. In this study, we successfully expressed FcaPV2 E6 and E7 putative oncogenes in feline epithelial cells and demonstrated that FcaPV2 E6 binds to p53, impairing its protein level. In addition, E6 and E7 inhibited ultraviolet B (UVB)-triggered accumulation of p53, p21 and pro-apoptotic markers such as Cleaved Caspase3, Bax and Bak, suggesting a synergistic action of the virus with UV exposure in tumour pathogenesis. Furthermore, FcaPV2 E7 bound to feline pRb and impaired pRb levels, resulting in upregulation of the downstream pro-proliferative genes Cyclin A and Cdc2. Importantly, we demonstrated mRNA expression of FcaPV2 E2, E6 and E7 in feline SCC samples, strengthening the hypothesis of a causative role in the development of feline SCC.
Asunto(s)
Carcinoma de Células Escamosas/etiología , Regulación Viral de la Expresión Génica , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/fisiología , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Activación Transcripcional , Animales , Proteínas Portadoras , Gatos , Línea Celular , Ratones , Infecciones por Papillomavirus/veterinaria , Unión Proteica , ARN Mensajero/genética , Transducción de SeñalRESUMEN
The beta genus of human papillomaviruses (ß-HPV) includes approximately 50 different viral types that are subdivided into five species (ß-1 through ß-5). Nonmelanoma cancers may involve some ß-1 and ß-2 HPV types, but the biology of most ß-HPV types and their possible connections to human disease are still little characterized. In this study, we studied the effects of ß-3 type HPV49 in a novel transgenic (Tg) mouse model, using a cytokeratin K14 promoter to drive expression of the E6 and E7 genes from this virus in the basal skin epidermis and the mucosal epithelia of the digestive tract (K14 HPV49 E6/E7-Tg mice). Viral oncogene expression only marginally increased cellular proliferation in the epidermis of Tg animals, compared with wild-type littermates, and we observed no spontaneous tumor formation during their entire lifespan. However, we found that K14 HPV49 E6/E7-Tg mice were highly susceptible to upper digestive tract carcinogenesis upon initiation with 4-nitroquinoline 1-oxide (4NQO). This was a selective effect, as the same mice did not exhibit any skin lesions after chronic UV irradiation. Opposite results were observed in an analogous Tg model expressing the ß-2 HPV38 E6 and E7 oncogenes at the same anatomic sites. While these mice were highly susceptible to UV-induced skin carcinogenesis, as previously shown, they were little affected by 4NQO treatment. Overall, our findings highlight important differences in the biologic properties of certain ß-type HPV that affect their impact on carcinogenesis in an anatomic site-specific manner. Cancer Res; 76(14); 4216-25. ©2016 AACR.
Asunto(s)
Neoplasias del Sistema Digestivo/etiología , Proteínas Oncogénicas Virales/fisiología , Papillomaviridae/patogenicidad , Proteínas E7 de Papillomavirus/fisiología , Animales , Carcinoma de Células Escamosas/etiología , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Transgénicos , Neoplasias Cutáneas/etiología , Rayos UltravioletaRESUMEN
Hepatitis C virus (HCV) is an oncogenic virus associated with the onset of hepatocellular carcinoma (HCC). The present study investigated the possible link between HCV infection and Netrin-1, a ligand for dependence receptors that sustains tumorigenesis, in particular in inflammation-associated tumors. We show that Netrin-1 expression is significantly elevated in HCV+ liver biopsies compared to hepatitis B virus (HBV+) and uninfected samples. Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues. Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression. In vitro, HCV increased the level and translation of Netrin-1 in a NS5A-La-related protein 1 (LARP1)-dependent fashion. Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells. Netrin-1 enhanced infectivity of HCV particles and promoted viral entry by increasing the activation and decreasing the recycling of the epidermal growth factor receptor (EGFR), a protein that is dysregulated in HCC. Netrin-1 and HCV are, therefore, reciprocal inducers in vitro and in patients, as seen from the increase in viral morphogenesis and viral entry, both phenomena converging toward an increase in the level of infectivity of HCV virions. This functional association involving a cancer-related virus and Netrin-1 argues for evaluating the implication of UNC5 receptor ligands in other oncogenic microbial species.
Asunto(s)
Receptores ErbB/metabolismo , Hepatitis C/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Autoantígenos/metabolismo , Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/metabolismo , Línea Celular , Transformación Celular Neoplásica , Hepatitis C/complicaciones , Hepatitis C/virología , Humanos , Cirrosis Hepática/metabolismo , Cirrosis Hepática/virología , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/metabolismo , Netrina-1 , Ribonucleoproteínas/metabolismo , Regulación hacia Arriba , Proteínas no Estructurales Virales/metabolismo , Internalización del Virus , Antígeno SS-BRESUMEN
Fusion to carrier proteins is an effective strategy for stabilizing and providing immunogenicity to peptide epitopes. This is commonly achieved by cross-linking of chemically synthesized peptides to carrier proteins. An alternative approach is internal grafting of selected peptide epitopes to a scaffold protein via double stranded-oligonucleotide insertion or gene synthesis, followed by recombinant expression of the resulting chimeric polypeptide. The scaffold protein should confer immunogenicity to the stabilized and structurally constrained peptide, but also afford easy production of the antigen in recombinant form. A macromolecular scaffold that meets the above criteria is the redox protein thioredoxin, especially bacterial thioredoxin. Here we describe our current methodology for internal grafting of selected peptide epitopes to thioredoxin as tandemly arranged multipeptide repeats ("Thioredoxin Displayed Multipeptide Immunogens"), bacterial expression and purification of the recombinant thioredoxin-multipeptide fusion proteins and their use as antigens for the production of anti-peptide antibodies for prophylactic vaccine as well as diagnostic purposes.
Asunto(s)
Antígenos/inmunología , Proteínas Portadoras , Epítopos/inmunología , Tiorredoxinas , Antígenos/química , Antígenos/genética , Antígenos/aislamiento & purificación , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Mapeo Epitopo , Epítopos/química , Epítopos/genética , Epítopos/aislamiento & purificación , Expresión Génica , Proteínas Recombinantes de Fusión , Tiorredoxinas/genética , Tiorredoxinas/inmunologíaRESUMEN
Although Epstein-Barr virus (EBV) infection is widely distributed, certain EBV-driven malignancies are geographically restricted. EBV-associated Burkitt's lymphoma (eBL) is endemic in children living in sub-Saharan Africa. This population is heavily exposed to food contaminated with the mycotoxin aflatoxin B1 (AFB1). Here, we show that exposure to AFB1 in in vitro and in vivo models induces activation of the EBV lytic cycle and increases EBV load, two events that are associated with an increased risk of eBL in vivo. AFB1 treatment leads to the alteration of cellular gene expression, with consequent activations of signaling pathways, e.g. PI3K, that in turn mediate reactivation of the EBV life cycle. Finally, we show that AFB1 triggers EBV-driven cellular transformation both in primary human B cells and in a humanized animal model. In summary, our data provide evidence for a role of AFB1 as a cofactor in EBV-mediated carcinogenesis.