RESUMEN
Etiologically, 5% of all cancers worldwide are caused by the high-risk human papillomaviruses (hrHPVs). These viruses encode two oncoproteins (E6 and E7) whose expression is required for cancer initiation and maintenance. Among their cellular targets are the p53 and the retinoblastoma tumor suppressor proteins. Inhibition of the hrHPV E6-mediated ubiquitylation of p53 through the E6AP ubiquitin ligase results in the stabilization of p53, leading to cellular apoptosis. We utilized a live cell high-throughput screen to determine whether exogenous microRNA (miRNA) transfection had the ability to stabilize p53 in hrHPV-positive cervical cancer cells expressing a p53-fluorescent protein as an in vivo reporter of p53 stability. Among the miRNAs whose transfection resulted in the greatest p53 stabilization was 375-3p, which has previously been reported to stabilize p53 in HeLa cells, providing validation of the screen. The top 32 miRNAs, in addition to 375-3p, were further assessed using a second cell-based p53 stability reporter system, as well as in nonreporter HeLa cells to examine their effects on endogenous p53 protein levels, resulting in the identification of 23 miRNAs whose transfection increased p53 levels in HeLa cells. While a few miRNAs that stabilized p53 led to decreases in E6AP protein levels, all targeted HPV oncoprotein expression. We further examined subsets of these miRNAs for their abilities to induce apoptosis and determined whether it was p53-mediated. The introduction of specific miRNAs revealed surprisingly heterogeneous responses in different cell lines. Nonetheless, some of the miRNAs described here have potential as therapeutics for treating HPV-positive cancers. IMPORTANCE Human papillomaviruses cause approximately 5% of all cancers worldwide and encode genes that contribute to both the initiation and maintenance of these cancers. The viral oncoprotein E6 is expressed in all HPV-positive cancers and functions by targeting the degradation of p53 through the engagement of the cellular ubiquitin ligase E6AP. Inhibiting the degradation of p53 leads to apoptosis in HPV-positive cancer cells. Using a high-throughput live cell assay, we identified several miRNAs whose transfection stabilize p53 in HPV-positive cells. These miRNAs have the potential to be used in the treatment of HPV-positive cancers.
Asunto(s)
Alphapapillomavirus/metabolismo , MicroARNs/genética , Proteína p53 Supresora de Tumor/metabolismo , Alphapapillomavirus/genética , Apoptosis , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Estabilidad Proteica , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Approximately 5% of cancers are caused by high-risk human papillomaviruses. Although very effective preventive vaccines will reduce this cancer burden significantly over the next several decades, they have no therapeutic effect for those already infected and remaining at risk for malignant progression of hrHPV lesions. HPV-associated cancers are dependent upon the expression of the viral E6 and E7 oncogenes. The oncogenic function of hrHPV E6 relies partially on its ability to induce p53 degradation. Since p53 is generally wildtype in hrHPV-associated cancers, p53 stabilization arrests proliferation, induces apoptosis and/or results in senescence. Here we describe a live cell, image-based high-throughput screen to identify compounds that stabilize p53 and/or affect viability in HPV-positive cancer HeLa cells. We validate the robustness and potential of this screening assay by assessing the activities of approximately 6,500 known bioactive compounds, illustrating its capability to function as a platform to identify novel therapeutics for hrHPV.
Asunto(s)
Aurora Quinasas/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento/métodos , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Topoisomerasa/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Femenino , Células HeLa , Papillomavirus Humano 18/genética , Humanos , Proteínas Oncogénicas Virales/metabolismo , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/diagnóstico por imagen , Infecciones por Papillomavirus/patología , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/virologíaRESUMEN
Deregulation of the HECT ubiquitin ligase UBE3A/E6AP has been implicated in Angelman syndrome as well as autism spectrum disorders. We and others have previously identified the 26S proteasome as one of the major UBE3A-interacting protein complexes. Here, we characterize the interaction of UBE3A and the proteasomal subunit PSMD4 (Rpn10/S5a). We map the interaction to the highly conserved Zn2+-binding N-terminal (AZUL) domain of UBE3A, the integrity of which is crucial for binding to PSMD4. Interestingly, two Angelman syndrome point mutations that affect the AZUL domain show an impaired ability to bind PSMD4. Although not affecting the ubiquitin ligase or the estrogen receptor α-mediated transcriptional regulation activities, these AZUL domain mutations prevent UBE3A from stimulating the Wnt/ß-catenin signaling pathway. Taken together, our data indicate that impaired binding to the 26S proteasome and consequential deregulation of Wnt/ß-catenin signaling might contribute to the functional defect of these mutants in Angelman syndrome.
Asunto(s)
Síndrome de Angelman/enzimología , Mutación Puntual , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Zinc/metabolismo , Síndrome de Angelman/genética , Humanos , Complejo de la Endopetidasa Proteasomal/genética , Proteínas de Unión al ARN , Ubiquitina-Proteína Ligasas/metabolismo , Vía de Señalización WntRESUMEN
The papillomavirus E2 protein executes numerous essential functions related to viral transcription, replication of viral DNA, and viral genome maintenance. Because E2 lacks enzymatic activity, many of these functions are mediated by interactions with host cellular proteins. Unbiased proteomics approaches have successfully identified a number of E2-host protein interactions. We have extended such studies and have identified and validated the cellular proteins structural maintenance of chromosome 5 (SMC5) and SMC6 as interactors of the viral E2 protein. These two proteins make up the core components of the SMC5/6 complex. The SMC5/6 complex is a member of the conserved structural maintenance of chromosomes (SMC) family of proteins, which are essential for genome maintenance. We have examined the role of SMC5/6 in various E2 functions. Our data suggest that SMC6 is not required for E2-mediated transcriptional activation, E1/E2-mediated transient replication, or differentiation-dependent amplification of viral DNA. Our data, however, suggest a role for SMC5/6 in viral genome maintenance.IMPORTANCE The high-risk human papillomaviruses (HPVs) are the etiological cause of cervical cancer and the most common sexually transmitted infection. While the majority of infections may be asymptomatic or cause only benign lesions, persistent infection with the oncogenic high-risk HPV types may lead to serious diseases, such as cervical cancer, anogenital carcinoma, or head and neck oropharyngeal squamous cell carcinoma. The identification of virus-host protein interactions provides insights into the mechanisms of viral DNA persistence, viral genome replication, and cellular transformation. Elucidating the mechanism of early events in the virus replication cycle as well as of integration of viral DNA into host chromatin may present novel antiviral strategies and targets for counteracting persistent infection. The E2 protein is an important viral regulatory protein whose functions are mediated through interactions with host cell proteins. Here we explore the interaction of E2 with SMC5/6 and the functional consequences.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/fisiología , Línea Celular Tumoral , Replicación del ADN , Células HEK293 , Humanos , Papillomaviridae/genética , Proteómica , Activación Transcripcional , Replicación ViralRESUMEN
Perturbations in activity and dosage of the UBE3A ubiquitin-ligase have been linked to Angelman syndrome and autism spectrum disorders. UBE3A was initially identified as the cellular protein hijacked by the human papillomavirus E6 protein to mediate the ubiquitylation of p53, a function critical to the oncogenic potential of these viruses. Although a number of substrates have been identified, the normal cellular functions and pathways affected by UBE3A are largely unknown. Previously, we showed that UBE3A associates with HERC2, NEURL4, and MAPK6/ERK3 in a high-molecular-weight complex of unknown function that we refer to as the HUN complex (HERC2, UBE3A, and NEURL4). In this study, the combination of two complementary proteomic approaches with a rigorous network analysis revealed cellular functions and pathways in which UBE3A and the HUN complex are involved. In addition to finding new UBE3A-associated proteins, such as MCM6, SUGT1, EIF3C, and ASPP2, network analysis revealed that UBE3A-associated proteins are connected to several fundamental cellular processes including translation, DNA replication, intracellular trafficking, and centrosome regulation. Our analysis suggests that UBE3A could be involved in the control and/or integration of these cellular processes, in some cases as a component of the HUN complex, and also provides evidence for crosstalk between the HUN complex and CAMKII interaction networks. This study contributes to a deeper understanding of the cellular functions of UBE3A and its potential role in pathways that may be affected in Angelman syndrome, UBE3A-associated autism spectrum disorders, and human papillomavirus-associated cancers.
Asunto(s)
Mapeo de Interacción de Proteínas , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Línea Celular , Células HEK293 , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
Retinoic acid-inducible gene I (RIG-I) is a key pattern recognition receptor that senses viral RNA and interacts with the mitochondrial adaptor MAVS, triggering a signaling cascade that results in the production of type I interferons (IFNs). This signaling axis is initiated by K63-linked ubiquitination of RIG-I mediated by the E3 ubiquitin ligase TRIM25, which promotes the interaction of RIG-I with MAVS. USP15 was recently identified as an upstream regulator of TRIM25, stabilizing the enzyme through removal of degradative K48-linked polyubiquitin, ultimately promoting RIG-I-dependent cytokine responses. Here, we show that the E6 oncoprotein of human papillomavirus type 16 (HPV16) as well as of other HPV types form a complex with TRIM25 and USP15 in human cells. In the presence of E6, the K48-linked ubiquitination of TRIM25 was markedly increased, and in line with this, TRIM25 degradation was enhanced. Our results further showed that E6 inhibited the TRIM25-mediated K63-linked ubiquitination of RIG-I and its CARD-dependent interaction with MAVS. HPV16 E6, but not E7, suppressed the RIG-I-mediated induction of IFN-ß, chemokines, and IFN-stimulated genes (ISGs). Finally, CRISPR-Cas9 gene targeting in human keratinocytes showed that the TRIM25-RIG-I-MAVS triad is important for eliciting an antiviral immune response to HPV16 infection. Our study thus identifies a novel immune escape mechanism that is conserved among different HPV strains and further indicates that the RIG-I signaling pathway plays an important role in the innate immune response to HPV infection.IMPORTANCE Persistent infection and tumorigenesis by HPVs are known to require viral manipulation of a variety of cellular processes, including those involved in innate immune responses. Here, we show that the HPV E6 oncoprotein antagonizes the activation of the cytoplasmic innate immune sensor RIG-I by targeting its upstream regulatory enzymes TRIM25 and USP15. We further show that the RIG-I signaling cascade is important for an antiviral innate immune response to HPV16 infection, providing evidence that RIG-I, whose role in sensing RNA virus infections has been well characterized, also plays a crucial role in the antiviral host response to small DNA viruses of the Papillomaviridae family.
Asunto(s)
Proteína 58 DEAD Box/inmunología , Papillomavirus Humano 6/inmunología , Inmunidad Innata , Queratinocitos/inmunología , Proteínas Oncogénicas Virales/inmunología , Infecciones por Papillomavirus/inmunología , Transducción de Señal/inmunología , Factores de Transcripción/inmunología , Proteínas de Motivos Tripartitos/inmunología , Ubiquitina-Proteína Ligasas/inmunología , Proteasas Ubiquitina-Específicas/inmunología , Proteína 58 DEAD Box/genética , Células HEK293 , Papillomavirus Humano 6/genética , Humanos , Queratinocitos/patología , Queratinocitos/virología , Proteínas Oncogénicas Virales/genética , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/patología , Receptores Inmunológicos , Transducción de Señal/genética , Factores de Transcripción/genética , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética , Proteasas Ubiquitina-Específicas/genéticaRESUMEN
Merkel cell carcinoma (MCC) frequently contains integrated copies of Merkel cell polyomavirus DNA that express a truncated form of Large T antigen (LT) and an intact Small T antigen (ST). While LT binds RB and inactivates its tumor suppressor function, it is less clear how ST contributes to MCC tumorigenesis. Here we show that ST binds specifically to the MYC homolog MYCL (L-MYC) and recruits it to the 15-component EP400 histone acetyltransferase and chromatin remodeling complex. We performed a large-scale immunoprecipitation for ST and identified co-precipitating proteins by mass spectrometry. In addition to protein phosphatase 2A (PP2A) subunits, we identified MYCL and its heterodimeric partner MAX plus the EP400 complex. Immunoprecipitation for MAX and EP400 complex components confirmed their association with ST. We determined that the ST-MYCL-EP400 complex binds together to specific gene promoters and activates their expression by integrating chromatin immunoprecipitation with sequencing (ChIP-seq) and RNA-seq. MYCL and EP400 were required for maintenance of cell viability and cooperated with ST to promote gene expression in MCC cell lines. A genome-wide CRISPR-Cas9 screen confirmed the requirement for MYCL and EP400 in MCPyV-positive MCC cell lines. We demonstrate that ST can activate gene expression in a EP400 and MYCL dependent manner and this activity contributes to cellular transformation and generation of induced pluripotent stem cells.
Asunto(s)
Antígenos Virales de Tumores/metabolismo , Carcinoma de Células de Merkel/virología , Transformación Celular Viral/fisiología , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Antígenos Transformadores de Poliomavirus/metabolismo , Carcinoma de Células de Merkel/genética , Carcinoma de Células de Merkel/metabolismo , Línea Celular Tumoral , Humanos , Immunoblotting , Inmunoprecipitación , Poliomavirus de Células de Merkel , Infecciones por Polyomavirus/complicaciones , Infecciones por Polyomavirus/genética , Infecciones por Polyomavirus/metabolismo , Infecciones Tumorales por Virus/complicaciones , Infecciones Tumorales por Virus/genética , Infecciones Tumorales por Virus/metabolismoRESUMEN
UNLABELLED: The major transformation activity of the high-risk human papillomaviruses (HPV) is associated with the E7 oncoprotein. The interaction of HPV E7 with retinoblastoma family proteins is important for several E7 activities; however, this interaction does not fully account for the high-risk E7-specific cellular immortalization and transformation activities. We have determined that the cellular non-receptor protein tyrosine phosphatase PTPN14 interacts with HPV E7 from many genus alpha and beta HPV types. We find that high-risk genus alpha HPV E7, but not low-risk genus alpha or beta HPV E7, is necessary and sufficient to reduce the steady-state level of PTPN14 in cells. High-risk E7 proteins target PTPN14 for proteasome-mediated degradation, which requires the ubiquitin ligase UBR4, and PTPN14 is degraded by the proteasome in HPV-positive cervical cancer cell lines. Residues in the C terminus of E7 interact with the C-terminal phosphatase domain of PTPN14, and interference with the E7-PTPN14 interaction restores PTPN14 levels in cells. Finally, PTPN14 degradation correlates with the retinoblastoma-independent transforming activity of high-risk HPV E7. IMPORTANCE: High-risk human papillomaviruses (HPV) are the cause of cervical cancer, some other anogenital cancers, and a growing fraction of oropharyngeal carcinomas. The high-risk HPV E6 and E7 oncoproteins enable these viruses to cause cancer, and the mechanistic basis of their carcinogenic activity has been the subject of intense study. The high-risk E7 oncoprotein is especially important in the immortalization and transformation of human cells, which makes it a central component of HPV-associated cancer development. E7 oncoproteins interact with retinoblastoma family proteins, but for several decades, it has been recognized that high-risk HPV E7 oncoproteins have additional cancer-associated activities. We have determined that high-risk E7 proteins target the proteolysis of the cellular protein tyrosine phosphatase PTPN14 and find that this activity is correlated with the retinoblastoma-independent transforming activity of E7.
Asunto(s)
Transformación Celular Viral , Papillomavirus Humano 16/patogenicidad , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Proteolisis , Proteínas de Unión a Calmodulina/metabolismo , Línea Celular Tumoral , Transformación Celular Viral/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Papillomavirus Humano 16/química , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina-Proteína Ligasas , Neoplasias del Cuello Uterino/virologíaRESUMEN
The role of infectious agents in cancer is generally underappreciated. However, approximately 20% of human cancers are caused by infectious agents and as such they rank second only to tobacco as a potentially preventable cause in humans. Specific viruses, parasites, and bacteria have been linked to specific human cancers. The infectious etiology for these specific cancers provides opportunities for prevention and treatment.
Asunto(s)
Antiinfecciosos/uso terapéutico , Infecciones Bacterianas/terapia , Neoplasias/prevención & control , Enfermedades Parasitarias/terapia , Infecciones Tumorales por Virus/terapia , Animales , Bacterias/efectos de los fármacos , Bacterias/patogenicidad , Infecciones Bacterianas/complicaciones , Infecciones Bacterianas/diagnóstico , Infecciones Bacterianas/microbiología , Transformación Celular Viral , Interacciones Huésped-Patógeno , Humanos , Neoplasias/diagnóstico , Neoplasias/etiología , Parásitos/efectos de los fármacos , Parásitos/patogenicidad , Enfermedades Parasitarias/complicaciones , Enfermedades Parasitarias/diagnóstico , Enfermedades Parasitarias/parasitología , Factores de Riesgo , Resultado del Tratamiento , Infecciones Tumorales por Virus/complicaciones , Infecciones Tumorales por Virus/diagnóstico , Infecciones Tumorales por Virus/virología , Virus/efectos de los fármacos , Virus/patogenicidadRESUMEN
A role for the beta genus HPVs in keratinocyte carcinoma (KC) remains to be established. In this article we examine the potential role of the beta HPVs in cancer revealed by the epidemiology associating these viruses with KC and supported by oncogenic properties of the beta HPV proteins. Unlike the cancer associated alpha genus HPVs, in which transcriptionally active viral genomes are invariably found associated with the cancers, that is not the case for the beta genus HPVs and keratinocyte carcinomas. Thus a role for the beta HPVs in KC would necessarily be in the carcinogenesis initiation and not in the maintenance of the tumor.
Asunto(s)
Betapapillomavirus/fisiología , Betapapillomavirus/patogenicidad , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/virología , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/virología , Carcinogénesis , Humanos , Queratinocitos/virología , Infecciones por Papillomavirus/epidemiología , Neoplasias Cutáneas/epidemiologíaRESUMEN
UNLABELLED: Many of the small DNA tumor viruses encode transforming proteins that function by targeting critical cellular pathways involved in cell proliferation and survival. In this study, we have examined whether some of the functions of the polyomavirus small T antigens (ST) are shared by the E6 and E7 oncoproteins of two oncogenic papillomaviruses. Using three different assays, we have found that E7 can provide some simian virus 40 (SV40) or murine polyomavirus (PyV) ST functions. Both human papillomavirus 16 (HPV16) and bovine papillomavirus (BPV1) E7 proteins are capable of partially substituting for SV40 ST in a transformation assay that also includes SV40 large T antigen, the catalytic subunit of cellular telomerase, and oncogenic Ras. Like SV40 ST, HPV16 E7 has the ability to override a quiescence block induced by mitogen deprivation. Like PyV ST, it also has the ability to inhibit myoblast differentiation. At least two of these activities are dependent upon the interaction of HPV16 E7 with retinoblastoma protein family members. For small T antigens, interaction with PP2A is needed for each of these functions. Even though there is no strong evidence that E6 or E7 share the ability of small T to interact with PP2A, E7 provides these functions related to cellular transformation. IMPORTANCE: DNA tumor viruses have provided major insights into how cancers develop. Some viruses, like the human papillomaviruses, can cause cancer directly. Both the papillomaviruses and the polyomaviruses have served as tools for understanding pathways that are often perturbed in cancer. Here, we have compared the functions of transforming proteins from several DNA tumor viruses, including two papillomaviruses and two polyomaviruses. We tested the papillomavirus E6 and E7 oncoproteins in three functional assays and found that E7 can provide some or all of the functions of the SV40 small T antigen, another well-characterized oncoprotein, in two of these assays. In a third assay, papillomavirus E7 has the same effect as the murine polyomavirus small T protein. In summary, we report several new functions for the papillomavirus E7 proteins, which will contribute new insights into the roles of viruses in cancer and the cellular pathways they perturb in carcinogenesis.
Asunto(s)
Antígenos Transformadores de Poliomavirus/metabolismo , Transformación Celular Viral , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Represoras/metabolismo , Prueba de Complementación Genética , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/fisiología , Humanos , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/fisiología , Virus 40 de los Simios/genética , Virus 40 de los Simios/fisiologíaRESUMEN
HERC2 is a large E3 ubiquitin ligase with multiple structural domains that has been implicated in an array of cellular processes. Mutations in HERC2 are linked to developmental delays and impairment caused by nervous system dysfunction, such as Angelman Syndrome and autism-spectrum disorders. However, HERC2 cellular activity and regulation remain poorly understood. We used a broad proteomic approach to survey the landscape of cellular proteins that interact with HERC2. We identified nearly 300 potential interactors, a subset of which we validated binding to HERC2. The potential HERC2 interactors included the eukaryotic translation initiation factor 3 complex, the intracellular transport COPI coatomer complex, the glycogen regulator phosphorylase kinase, beta-catenin, PI3 kinase, and proteins involved in fatty acid transport and iron homeostasis. Through a complex bioinformatic analysis of potential interactors, we linked HERC2 to cellular processes including intracellular protein trafficking and transport, metabolism of cellular energy, and protein translation. Given its size, multidomain structure, and association with various cellular activities, HERC2 may function as a scaffold to integrate protein complexes and bridge critical cellular pathways. This work provides a significant resource with which to interrogate HERC2 function more deeply and evaluate its contributions to mechanisms governing cellular homeostasis and disease.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteoma/análisis , Proteoma/metabolismo , Factores de Intercambio de Guanina Nucleótido/análisis , Humanos , Proteínas/análisis , Proteínas/metabolismo , Proteínas/fisiología , Proteómica , Ubiquitina-Proteína LigasasRESUMEN
UNLABELLED: Several recent studies have converged upon the innate immune DNA cytosine deaminase APOBEC3B (A3B) as a significant source of genomic uracil lesions and mutagenesis in multiple human cancers, including those of the breast, head/neck, cervix, bladder, lung, ovary, and other tissues. A3B is upregulated in these tumor types relative to normal tissues, but the mechanism is unclear. Because A3B also has antiviral activity in multiple systems and is a member of the broader innate immune response, we tested the hypothesis that human papillomavirus (HPV) infection causes A3B upregulation. We found that A3B mRNA expression and enzymatic activity were upregulated following transfection of a high-risk HPV genome and that this effect was abrogated by inactivation of E6. Transduction experiments showed that the E6 oncoprotein alone was sufficient to cause A3B upregulation, and a panel of high-risk E6 proteins triggered higher A3B levels than did a panel of low-risk or noncancer E6 proteins. Knockdown experiments in HPV-positive cell lines showed that endogenous E6 is required for A3B upregulation. Analyses of publicly available head/neck cancer data further support this relationship, as A3B levels are higher in HPV-positive cancers than in HPV-negative cancers. Taken together with the established role for high-risk E6 in functional inactivation of TP53 and published positive correlations in breast cancer between A3B upregulation and genetic inactivation of TP53, our studies suggest a model in which high-risk HPV E6, possibly through functional inactivation of TP53, causes derepression of A3B gene transcription. This would lead to a mutator phenotype that explains the observed cytosine mutation biases in HPV-positive head/neck and cervical cancers. IMPORTANCE: The innate immune DNA cytosine deaminase APOBEC3B (A3B) accounts for a large proportion of somatic mutations in cervical and head/neck cancers, but nothing is known about the mechanism responsible for its upregulation in these tumor types. Almost all cervical carcinomas and large proportions of head/neck tumors are caused by human papillomavirus (HPV) infection. Here, we establish a mechanistic link between HPV infection and A3B upregulation. The E6 oncoprotein of high-risk, but not low-risk, HPV types triggers A3B upregulation, supporting a model in which TP53 inactivation causes a derepression of A3B gene transcription and elevated A3B enzyme levels. This virus-induced mutator phenotype provides a mechanistic explanation for A3B signature mutations observed in HPV-positive head/neck and cervical carcinomas and may also help to account for the preferential cancer predisposition caused by high-risk HPV isolates.
Asunto(s)
Citidina Desaminasa/biosíntesis , Proteínas de Unión al ADN/metabolismo , Interacciones Huésped-Patógeno , Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/fisiología , Proteínas Represoras/metabolismo , Células Cultivadas , Humanos , Queratinocitos/enzimología , Queratinocitos/virología , Antígenos de Histocompatibilidad Menor , Mutación , Transducción Genética , Transfección , Regulación hacia ArribaRESUMEN
An important step in the malignant progression of HPV-associated lesions is the dysregulation of expression of the viral E6 and E7 oncogenes. This is often achieved through the loss of expression of E2, which represses the HPV LCR promoter and E6/E7 expression. Our previous studies confirmed a role for Brd4 in mediating the E2 transcriptional repression function, and identified JARID1C/SMCX and EP400 as contributors to E2-mediated repression. Here we show that TIP60, a component of the TIP60/TRRAP histone acetyltransferase complex, also contributes to the E2 repression function, and we extend our studies on SMCX. Di- and tri-methyl marks on histone H3K4 are reduced in the presence of E2 and SMCX, suggesting a mechanism by which SMCX contributes to E2-mediated repression of the HPV LCR. Together, these findings lead us to hypothesize that E2 recruits histone-modifying cellular proteins to the HPV LCR, resulting in transcriptional repression of E6 and E7.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Histona Acetiltransferasas/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Proteínas de Ciclo Celular , Línea Celular , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/fisiología , Histona Acetiltransferasas/genética , Histona Demetilasas , Humanos , Lisina Acetiltransferasa 5 , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas Virales/genética , Oxidorreductasas N-Desmetilantes/genética , Plásmidos , Regiones Promotoras Genéticas , Unión Proteica , Subunidades de Proteína , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
UNLABELLED: NUT midline carcinoma (NMC) is an aggressive subtype of squamous cell carcinoma that typically harbors BRD4/3-NUT fusion oncoproteins that block differentiation and maintain tumor growth. In 20% of cases, NUT is fused to uncharacterized non-BRD gene(s). We established a new patient-derived NMC cell line (1221) and demonstrated that it harbors a novel NSD3-NUT fusion oncogene. We find that NSD3-NUT is both necessary and sufficient for the blockade of differentiation and maintenance of proliferation in NMC cells. NSD3-NUT binds to BRD4, and BRD bromodomain inhibitors induce differentiation and arrest proliferation of 1221 cells. We find further that NSD3 is required for the blockade of differentiation in BRD4-NUT-expressing NMCs. These findings identify NSD3 as a novel critical oncogenic component and potential therapeutic target in NMC. SIGNIFICANCE: The existence of a family of fusion oncogenes in squamous cell carcinoma is unprecedented, and should lead to key insights into aberrant differentiation in NMC and possibly other squamous cell carcinomas. The involvement of the NSD3 methyltransferase as a component of the NUT fusion protein oncogenic complex identifies a new potential therapeutic target.
Asunto(s)
Carcinoma de Células Escamosas/genética , N-Metiltransferasa de Histona-Lisina/genética , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/genética , Proteínas Oncogénicas/genética , Adolescente , Carcinogénesis/genética , Carcinoma de Células Escamosas/patología , Proteínas de Ciclo Celular , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Humanos , Proteínas de Neoplasias , Proteínas Nucleares/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Factores de Transcripción/genéticaRESUMEN
UNLABELLED: The genus beta human papillomaviruses (beta HPVs) cause cutaneous lesions and are thought to be involved in the initiation of some nonmelanoma skin cancers (NMSCs), particularly in patients with the genetic disorder epidermodysplasia verruciformis (EV). We have previously reported that at least two of the genus beta HPV E6 proteins bind to and/or increase the steady-state levels of p53 in squamous epithelial cells. This is in contrast to a well-characterized ability of the E6 proteins of cancer-associated HPVs of genus alpha HPV, which inactivate p53 by targeting its ubiquitin-mediated proteolysis. In this study, we have investigated the ability of genus beta E6 proteins from eight different HPV types to block the transactivation of p53 target genes following DNA damage. We find that the E6 proteins from diverse beta HPV species and types vary in their capacity to block the induction of MDM2, p21, and proapoptotic genes after genotoxic stress. We conclude that some genus beta HPV E6 proteins inhibit at least some p53 target genes, although perhaps not by the same mechanism or to the same degree as the high-risk genus alpha HPV E6 proteins. IMPORTANCE: This study addresses the ability of various human papillomavirus E6 proteins to block the activation of p53-responsive cellular genes following DNA damage in human keratinocytes, the normal host cell for HPVs. The E6 proteins encoded by the high-risk, cancer-associated HPV types of genus alpha HPV have a well-established activity to target p53 degradation and thereby inhibit the response to DNA damage. In this study, we have investigated the ability of genus beta HPV E6 proteins from eight different HPV types to block the ability of p53 to transactivate downstream genes following DNA damage. We find that some, but not all, genus beta HPV E6 proteins can block the transactivation of some p53 target genes. This differential response to DNA damage furthers the understanding of cutaneous HPV biology and may help to explain the potential connection between some beta HPVs and cancer.
Asunto(s)
Betapapillomavirus/fisiología , Interacciones Huésped-Patógeno , Proteínas Oncogénicas Virales/metabolismo , Activación Transcripcional , Apoptosis , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Daño del ADN , Humanos , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidoresRESUMEN
The identification of interactions between viral and host cellular proteins has provided major insights into papillomavirus research, and these interactions are especially relevant to the role of papillomaviruses in the cancers with which they are associated. Recent advances in mass spectrometry technology and data processing now allow the systematic identification of such interactions. This has led to an improved understanding of the different pathologies associated with the many papillomavirus types, and the diverse nature of these viruses is reflected in the spectrum of interactions with host proteins. Here we review a history of proteomic approaches, particularly as applied to the papillomaviruses, and summarize current techniques. Current proteomic studies on the papillomaviruses use yeast-two-hybrid or affinity purification-mass spectrometry approaches. We detail the advantages and disadvantages of each and describe current examples of papillomavirus proteomic studies, with a particular focus on the HPV E6 and E7 oncoproteins.
Asunto(s)
Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/genética , Papillomaviridae/inmunología , Proteínas E7 de Papillomavirus/metabolismo , Infecciones por Papillomavirus/metabolismo , Proteoma/metabolismo , Sitios de Unión , Variación Genética/inmunología , Interacciones Huésped-Patógeno , Humanos , Espectrometría de Masas , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/genética , Papillomaviridae/clasificación , Proteínas E7 de Papillomavirus/química , Proteínas E7 de Papillomavirus/genética , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/virología , Filogenia , Unión Proteica , Mapeo de Interacción de Proteínas , Técnicas del Sistema de Dos HíbridosRESUMEN
We have begun to define the human papillomavirus (HPV)-associated proteome for a subset of the more than 120 HPV types that have been identified to date. Our approach uses a mass spectrometry-based platform for the systematic identification of interactions between human papillomavirus and host cellular proteins, and here we report a proteomic analysis of the E6 proteins from 16 different HPV types. The viruses included represent high-risk, low-risk, and non-cancer-associated types from genus alpha as well as viruses from four different species in genus beta. The E6 interaction data set consists of 153 cellular proteins, including several previously reported HPV E6 interactors such as p53, E6AP, MAML1, and p300/CBP and proteins containing PDZ domains. We report the genus-specific binding of E6s to either E6AP or MAML1, define the specific HPV E6s that bind to p300, and demonstrate several new features of interactions involving beta HPV E6s. In particular, we report that several beta HPV E6s bind to proteins containing PDZ domains and that at least two beta HPV E6s bind to p53. Finally, we report the newly discovered interaction of proteins of E6 of beta genus, species 2, with the Ccr4-Not complex, the first report of a viral protein binding to this complex. This data set represents a comprehensive survey of E6 binding partners that provides a resource for the HPV field and will allow continued studies on the diverse biology of the human papillomaviruses.
Asunto(s)
Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/metabolismo , Secuencia de Aminoácidos , Línea Celular , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Proteínas Oncogénicas Virales/química , Sistemas de Lectura Abierta , Papillomaviridae/clasificación , Unión Proteica , Homología de Secuencia de AminoácidoRESUMEN
The Notch signaling pathway is a key determinant in keratinocyte differentiation and growth cycle arrest, and has been reported to have a tumor suppressor function in skin. The papillomavirus life cycle is intricately linked to the differentiation status of keratinocytes. Papillomaviruses are associated with benign proliferative epithelial lesions in their respective hosts. Although human papillomaviruses (HPVs) associated with genital tract lesions have been extensively studied, studies of the cutaneous HPVs are more limited. In particular, it is well established that the E6 proteins of high-risk HPVs of the α-genus such as HPV16 and HPV18 mediate the degradation of p53 by its association with the ubiquitin ligase E6AP. In contrast, less is known about the cellular activities of the cutaneous HPVs of the ß-genus. By using an unbiased proteomic approach, we identify MAML1 and other members of the Notch transcription complex as high-confidence cellular interacting proteins of E6 proteins of the ß-genus HPVs and of the bovine papillomavirus type 1 associated with cutaneous fibropapillomas. We show that bovine papillomavirus type 1 and ß-HPV E6 repress Notch transcriptional activation, and that this repression is dependent on an interaction with MAML1. Finally, we show that the expression levels of endogenous Notch target genes are repressed by ß-HPV E6 proteins. These findings elucidate a mechanism of viral antagonism of Notch signaling, and suggest that Notch signaling is an important epithelial cell pathway target for the ß-HPVs.