RESUMO
Merkel cell polyomavirus (MCV) is a small DNA tumor virus that persists in human skin and causes Merkel cell carcinoma (MCC) in immunocompromised individuals. The multi-functional protein MCV small T (sT) activates viral DNA replication by stabilizing large T (LT) and promotes cell transformation through the LT stabilization domain (LTSD). Using MCVΔsT, a mutant MCV clone that ablates sT, we investigated the role of sT in MCV genome maintenance. sT was dispensable for initiation of viral DNA replication, but essential for maintenance of the MCV genome and activation of viral early and late gene expression for progression of the viral lifecycle. Furthermore, in phenotype rescue studies, exogenous sT activated viral DNA replication and mRNA expression in MCVΔsT through the LTSD. While exogenous LT expression, which mimics LT stabilization, increased viral DNA replication, it did not activate viral mRNA expression. After cataloging transcriptional regulator proteins by proximity-based MCV sT-host protein interaction analysis, we validated LTSD-dependent sT interaction with four transcriptional regulators: Cux1, c-Jun, BRD9, and CBP. Functional studies revealed Cux1 and c-Jun as negative regulators, and CBP and BRD9 as positive regulators of MCV transcription. CBP inhibitor A-485 suppressed sT-induced viral gene activation in replicating MCVΔsT and inhibited early gene expression in MCV-integrated MCC cells. These results suggest that sT promotes viral lifecycle progression by activating mRNA expression and capsid protein production through interaction with the transcriptional regulators. This activity is essential for MCV genome maintenance, suggesting a critical role of sT in MCV persistence and MCC carcinogenesis.
Assuntos
Carcinoma de Célula de Merkel , Poliomavírus das Células de Merkel , Infecções por Polyomavirus , Neoplasias Cutâneas , Infecções Tumorais por Vírus , Humanos , Poliomavírus das Células de Merkel/metabolismo , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/metabolismo , Transcrição Viral , Replicação do DNA , Replicação Viral , DNA Viral/genética , DNA Viral/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias Cutâneas/patologia , Genoma Viral , RNA Mensageiro/metabolismo , Infecções por Polyomavirus/metabolismoRESUMO
Merkel cell carcinoma (MCC) is a highly aggressive skin cancer associated with integration of Merkel cell polyomavirus (MCPyV). MCPyV-encoded T-antigens (TAs) are pivotal for sustaining MCC's oncogenic phenotype, i.e., repression of TAs results in reactivation of the RB pathway and subsequent cell cycle arrest. However, the MCC cell line LoKe, characterized by a homozygous loss of the RB1 gene, exhibits uninterrupted cell cycle progression after shRNA-mediated TA repression. This unique feature allows an in-depth analysis of the effects of TAs beyond inhibition of the RB pathway, revealing the decrease in expression of stem cell-related genes upon panTA-knockdown. Analysis of gene regulatory networks identified members of the E2F family (E2F1, E2F8, TFDP1) as key transcriptional regulators that maintain stem cell properties in TA-expressing MCC cells. Furthermore, minichromosome maintenance (MCM) genes, which encodes DNA-binding licensing proteins essential for stem cell maintenance, were suppressed upon panTA-knockdown. The decline in stemness occurred simultaneously with neural differentiation, marked by the increased expression of neurogenesis-related genes such as neurexins, BTG2, and MYT1L. This upregulation can be attributed to heightened activity of PBX1 and BPTF, crucial regulators of neurogenesis pathways. The observations in LoKe were confirmed in an additional MCPyV-positive MCC cell line in which RB1 was silenced before panTA-knockdown. Moreover, spatially resolved transcriptomics demonstrated reduced TA expression in situ in a part of a MCC tumor characterized by neural differentiation. In summary, TAs are critical for maintaining stemness of MCC cells and suppressing neural differentiation, irrespective of their impact on the RB-signaling pathway.
Assuntos
Antígenos Transformantes de Poliomavirus , Antígenos Virais de Tumores , Poliomavírus das Células de Merkel , Células-Tronco Neoplásicas , Proteínas de Ligação a Retinoblastoma , Humanos , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/metabolismo , Carcinoma de Célula de Merkel/virologia , Carcinoma de Célula de Merkel/genética , Diferenciação Celular , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Poliomavírus das Células de Merkel/genética , Células-Tronco Neoplásicas/virologia , Células-Tronco Neoplásicas/metabolismo , Neurônios/virologia , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Neoplasias Cutâneas/virologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismoRESUMO
SARS-CoV-2 NSP12, the viral RNA-dependent RNA polymerase (RdRp), is required for viral replication and is a therapeutic target to treat COVID-19. To facilitate research on SARS-CoV-2 NSP12 protein, we developed a rat monoclonal antibody (CM12.1) against the NSP12 N-terminus that can facilitate functional studies. Immunoblotting and immunofluorescence assay (IFA) confirmed the specific detection of NSP12 protein by this antibody for cells overexpressing the protein. Although NSP12 is generated from the ORF1ab polyprotein, IFA of human autopsy COVID-19 lung samples revealed NSP12 expression in only a small fraction of lung cells including goblet, club-like, vascular endothelial cells, and a range of immune cells, despite wide-spread tissue expression of spike protein antigen. Similar studies using in vitro infection also generated scant protein detection in cells with established virus replication. These results suggest that NSP12 may have diminished steady-state expression or extensive posttranslation modifications that limit antibody reactivity during SARS-CoV-2 replication.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Animais , Ratos , SARS-CoV-2/metabolismo , Anticorpos Monoclonais , Células Endoteliais , RNA Polimerase Dependente de RNA/genética , Antivirais/metabolismoRESUMO
The ongoing COVID-19 pandemic severely impacts global public health and economies. To facilitate research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virology and antiviral discovery, a noninfectious viral replicon system operating under biosafety level 2 containment is warranted. We report herein the construction and characterization of two SARS-CoV-2 minigenome replicon systems. First, we constructed the IVT-CoV2-Rep complementary DNA template to generate a replicon messenger RNA (mRNA) with nanoluciferase (NLuc) reporter via in vitro transcription (IVT). The replicon mRNA transfection assay demonstrated a rapid and transient replication of IVT-CoV2-Rep in a variety of cell lines, which could be completely abolished by known SARS-CoV-2 replication inhibitors. Our data also suggest that the transient phenotype of IVT-CoV2-Rep is not due to host innate antiviral responses. In addition, we have developed a DNA-launched replicon BAC-CoV2-Rep, which supports the in-cell transcription of a replicon mRNA as initial replication template. The BAC-CoV2-Rep transient transfection system exhibited a much stronger and longer replicon signal compared to the IVT-CoV2-Rep version. We also found that a portion of the NLuc reporter signal was derived from the spliced BAC-CoV2-Rep mRNA and was resistant to antiviral treatment, especially during the early phase after transfection. In summary, the established SARS-CoV-2 transient replicon systems are suitable for basic and antiviral research, and hold promise for stable replicon cell line development with further optimization.
Assuntos
COVID-19 , SARS-CoV-2 , Antivirais/farmacologia , Humanos , Pandemias , RNA Mensageiro , Replicon , SARS-CoV-2/genética , Replicação ViralRESUMO
Viral cancers show oncogene addiction to viral oncoproteins, which are required for survival and proliferation of the dedifferentiated cancer cell. Human Merkel cell carcinomas (MCCs) that harbor a clonally integrated Merkel cell polyomavirus (MCV) genome have low mutation burden and require viral T antigen expression for tumor growth. Here, we showed that MCV+ MCC cells cocultured with keratinocytes undergo neuron-like differentiation with neurite outgrowth, secretory vesicle accumulation, and the generation of sodium-dependent action potentials, hallmarks of a neuronal cell lineage. Cocultured keratinocytes are essential for induction of the neuronal phenotype. Keratinocyte-conditioned medium was insufficient to induce this phenotype. Single-cell RNA sequencing revealed that T antigen knockdown inhibited cell cycle gene expression and reduced expression of key Merkel cell lineage/MCC marker genes, including HES6, SOX2, ATOH1, and KRT20 Of these, T antigen knockdown directly inhibited Sox2 and Atoh1 expression. MCV large T up-regulated Sox2 through its retinoblastoma protein-inhibition domain, which in turn activated Atoh1 expression. The knockdown of Sox2 in MCV+ MCCs mimicked T antigen knockdown by inducing MCC cell growth arrest and neuron-like differentiation. These results show Sox2-dependent conversion of an undifferentiated, aggressive cancer cell to a differentiated neuron-like phenotype and suggest that the ontology of MCC arises from a neuronal cell precursor.
Assuntos
Antígenos Virais de Tumores/genética , Carcinoma de Célula de Merkel/etiologia , Carcinoma de Célula de Merkel/metabolismo , Poliomavírus das Células de Merkel/genética , Fenótipo , Infecções por Polyomavirus/complicações , Fatores de Transcrição SOXB1/genética , Antígenos Virais de Tumores/imunologia , Antígenos Virais de Tumores/metabolismo , Carcinoma de Célula de Merkel/patologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Linhagem da Célula/genética , Transformação Celular Viral , Técnicas de Silenciamento de Genes , Humanos , Queratinócitos , Células de Merkel/metabolismo , Poliomavírus das Células de Merkel/imunologia , Neuritos/metabolismo , Neurônios/metabolismo , Infecções por Polyomavirus/imunologia , Infecções por Polyomavirus/virologia , Fatores de Transcrição SOXB1/metabolismo , Infecções Tumorais por Vírus/complicações , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/virologiaRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Movimento Celular/genética , Proliferação de Células/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Poliomavírus das Células de Merkel/genética , Poliomavírus das Células de Merkel/fisiologia , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/metabolismo , Carcinogênese/genética , Carcinoma de Célula de Merkel/virologia , Linhagem Celular , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Humanos , Queratinócitos/virologia , Infecções por Polyomavirus/virologia , Pele/patologia , Neoplasias Cutâneas/genética , Transcriptoma , Infecções Tumorais por Vírus/virologiaRESUMO
Mammalian target of rapamycin (mTOR)-directed eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation promotes cap-dependent translation and tumorigenesis. During mitosis, cyclin-dependent kinase 1 (CDK1) substitutes for mTOR and fully phosphorylates 4E-BP1 at canonical sites (T37, T46, S65, and T70) and the noncanonical S83 site, resulting in a mitosis-specific hyperphosphorylated δ isoform. Colocalization studies with a phospho-S83 specific antibody indicate that 4E-BP1 S83 phosphorylation accumulates at centrosomes during prophase, peaks at metaphase, and decreases through telophase. Although S83 phosphorylation of 4E-BP1 does not affect general cap-dependent translation, expression of an alanine substitution mutant 4E-BP1.S83A partially reverses rodent cell transformation induced by Merkel cell polyomavirus small T antigen viral oncoprotein. In contrast to inhibitory mTOR 4E-BP1 phosphorylation, these findings suggest that mitotic CDK1-directed phosphorylation of δ-4E-BP1 may yield a gain of function, distinct from translation regulation, that may be important in tumorigenesis and mitotic centrosome function.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Quinase CDC2/metabolismo , Transformação Celular Neoplásica/metabolismo , Fosfoproteínas/metabolismo , Serina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Centrossomo/metabolismo , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Mitose/genética , Mutação , Fosfoproteínas/genética , Fosforilação , Biossíntese de Proteínas , RNA Mensageiro/genética , Serina/genéticaRESUMO
Trichodysplasia spinulosa-associated polyomavirus (TSPyV), a newly identified polyomavirus, has been implicated as a causative agent of trychodysplasia spinulosa (TS), a rare proliferative skin disease in severely immunocompromised hosts. Diagnosis using mAbs is a promising tool with high specificity towards the specific antigen. However, thus far, no suitable mAbs for diagnosing TS disease have been identified. In this study, mAbs specific for VP1 of TSPyV were developed and characterized. Wheat germ cell-free synthesized VP1 protein of TSPyV was used to immunize BALB/c mice to generate hybridomas. Screening of the resultant hybridoma clones resulted in selection of five strongly positive clones that produce mAbs that react with the TSPyV-VP1 antigen. Epitope mapping and bioinformatic analysis showed that these mAbs recognized epitopes located within highly conserved C-terminal region of all clinical isolates of TSPyV-VP1. Further, all these mAbs were highly effective for immunofluorescence and immunoprecipitation analysis. Three of the five mAbs exhibited no cross-reactivity with VP1 of other related polyomaviruses. In addition, one of our mAbs (#14) provided immunohistochemical staining of skin tissue of TS disease. It can be concluded that three of the mAbs in this panel of anti-VP1 antibodies may provide a useful set of tools for studying TSPyV infection and making the specific diagnosis.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Proteínas do Capsídeo/imunologia , Infecções por Polyomavirus/imunologia , Polyomavirus/imunologia , Infecções Tumorais por Vírus/imunologia , Animais , Proteínas do Capsídeo/genética , DNA Viral , Modelos Animais de Doenças , Mapeamento de Epitopos , Epitopos/imunologia , Feminino , Regulação Viral da Expressão Gênica , Genes Virais/genética , Humanos , Hibridomas , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Modelos Moleculares , Polyomavirus/genética , Infecções por Polyomavirus/diagnóstico , Infecções por Polyomavirus/virologia , Alinhamento de Sequência , Pele/patologia , Infecções Tumorais por Vírus/diagnósticoRESUMO
Mitosis is commonly thought to be associated with reduced cap-dependent protein translation. Here we show an alternative control mechanism for maintaining cap-dependent translation during mitosis revealed by a viral oncoprotein, Merkel cell polyomavirus small T (MCV sT). We find MCV sT to be a promiscuous E3 ligase inhibitor targeting the anaphase-promoting complex, which increases cell mitogenesis. MCV sT binds through its Large T stabilization domain region to cell division cycle protein 20 (Cdc20) and, possibly, cdc20 homolog 1 (Cdh1) E3 ligase adapters. This activates cyclin-dependent kinase 1/cyclin B1 (CDK1/CYCB1) to directly hyperphosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) at authentic sites, generating a mitosis-specific, mechanistic target of rapamycin (mTOR) inhibitor-resistant δ phospho-isoform not present in G1-arrested cells. Recombinant 4E-BP1 inhibits capped mRNA reticulocyte translation, which is partially reversed by CDK1/CYCB1 phosphorylation of 4E-BP1. eIF4G binding to the eIF4E-m(7)GTP cap complex is resistant to mTOR inhibition during mitosis but sensitive during interphase. Flow cytometry, with and without sT, reveals an orthogonal pH3(S10+) mitotic cell population having higher inactive p4E-BP1(T37/T46+) saturation levels than pH3(S10-) interphase cells. Using a Click-iT flow cytometric assay to directly measure mitotic protein synthesis, we find that most new protein synthesis during mitosis is cap-dependent, a result confirmed using the eIF4E/4G inhibitor drug 4E1RCat. For most cell lines tested, cap-dependent translation levels were generally similar between mitotic and interphase cells, and the majority of new mitotic protein synthesis was cap-dependent. These findings suggest that mitotic cap-dependent translation is generally sustained during mitosis by CDK1 phosphorylation of 4E-BP1 even under conditions of reduced mTOR signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos Transformantes de Poliomavirus/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Biossíntese de Proteínas , Serina-Treonina Quinases TOR/metabolismo , Proteína Quinase CDC2 , Proteínas de Ciclo Celular , Proliferação de Células , Células HEK293 , Células HeLa , Humanos , Interfase , Mitose , Nocodazol/química , Fosforilação , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
UNLABELLED: Merkel cell polyomavirus (MCV) is a newly discovered human cancer virus encoding a small T (sT) oncoprotein. We performed MCV sT FLAG-affinity purification followed by mass spectroscopy (MS) analysis, which identified several protein phosphatases (PP), including PP2A A and C subunits and PP4C, as potential cellular interacting proteins. PP2A targeting is critical for the transforming properties of nonhuman polyomaviruses, such as simian virus 40 (SV40), but is not required for MCV sT-induced rodent cell transformation. We compared similarities and differences in PP2A binding between MCV and SV40 sT. While SV40 sT coimmunopurified with subunits PP2A Aα and PP2A C, MCV sT coimmunopurified with PP2A Aα, PP2A Aß, and PP2A C. Scanning alanine mutagenesis at 29 sites across the MCV sT protein revealed that PP2A-binding domains lie on the opposite molecular surface from a previously described large T stabilization domain (LSD) loop that binds E3 ligases, such as Fbw7. MCV sT-PP2A interactions can be functionally distinguished by mutagenesis from MCV sT LSD-dependent 4E-BP1 hyperphosphorylation and viral DNA replication enhancement. MCV sT has a restricted range for PP2A B subunit substitution, inhibiting only the assembly of B56α into the phosphatase holoenzyme. In contrast, SV40 sT inhibits the assembly of B55α, B56α and B56ε into PP2A. We conclude that MCV sT is required for Merkel cell carcinoma growth, but its in vitro transforming activity depends on LSD interactions rather than PP2A targeting. IMPORTANCE: Merkel cell polyomavirus is a newly discovered human cancer virus that promotes cancer, in part, through expression of its small T (sT) oncoprotein. Animal polyomavirus sT oncoproteins have been found to cause experimental tumors by blocking the activities of a group of phosphatases called protein phosphatase 2A (PP2A). Our structural analysis reveals that MCV sT also displaces the B subunit of PP2A to inhibit PP2A activity. MCV sT, however, only displaces a restricted subset of PP2A B subunits, which is insufficient to cause tumor cell formation in vitro. MCV sT instead transforms tumor cells through another region called the large T stabilization domain. The PP2A targeting and transforming activities lie on opposite faces of the MCV sT molecule and can be genetically separated from each other.
Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Transformação Celular Neoplásica/metabolismo , Poliomavírus das Células de Merkel/metabolismo , Proteína Fosfatase 2/metabolismo , Antígenos Transformantes de Poliomavirus/genética , Cromatografia de Afinidade , Células HEK293 , Humanos , Immunoblotting , Imunoprecipitação , Espectrometria de Massas , Mutagênese , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Merkel cell polyomavirus (MCV) is clonally integrated in over 80 % of Merkel cell carcinomas and mediates tumour development through the expression of viral oncoproteins, the large T (LT) and small T antigens (sT). Viral integration is associated with signature mutations in the T-antigen locus that result in deletions of C-terminal replicative functions of the LT antigen. Despite these truncations, the LT LXCXE retinoblastoma (Rb) pocket protein family binding domain is retained, and the entire sT isoform is maintained intact. To investigate the ability of MCV oncoproteins to regulate host gene expression, we performed microarray analysis on cells stably expressing tumour-derived LT, tumour-derived LT along with sT, and tumour-derived LT with a mutated Rb interaction domain. Gene expression alterations in the presence of tumour-derived LT could be classified into three main groups: genes that are involved in the cell cycle (specifically the G1/S transition), genes involved in DNA replication and genes involved in cellular movement. The LXCXE mutant LT largely reversed gene expression alterations detected with the WT tumour-derived LT, while co-expression of sT did not significantly affect these patterns of gene expression. LXCXE-dependent upregulation of cyclin E and CDK2 correlated with increased proliferation in tumour-derived LT-expressing cells. Tumour-derived LT and tumour-derived LT plus sT increased expression of multiple cytokines and chemokines, which resulted in elevated levels of secreted IL-8. We concluded that, in human fibroblasts, the LXCXE motif of tumour-derived LT enhances cellular proliferation and upregulates cell cycle and immune signalling gene transcription.
Assuntos
Antígenos Virais de Tumores/fisiologia , Citocinas/metabolismo , Regulação da Expressão Gênica/imunologia , Inflamação/metabolismo , Poliomavírus das Células de Merkel/imunologia , Antígenos Virais de Tumores/imunologia , Proliferação de Células , Células Cultivadas , Citocinas/genética , Reparo do DNA , Replicação do DNA/fisiologia , Fibroblastos/fisiologia , Fibroblastos/virologia , Regulação da Expressão Gênica/fisiologia , Humanos , Transdução de Sinais/imunologia , Transcrição Gênica , Regulação para CimaRESUMO
Establishment of a chronic infection is a key event in virus-mediated carcinogenesis. Several cancer-associated, double-stranded DNA (dsDNA) viruses act via their oncoproteins to downregulate Toll-like receptor 9 (TLR9), a key receptor in the host innate immune response that senses viral or bacterial dsDNA. A novel oncogenic virus, Merkel cell polyomavirus (MCPyV), has been recently identified that causes up to 80% of Merkel cell carcinomas (MCCs). However, it is not yet known whether this oncogenic virus also disrupts immune-related pathways. We find that MCPyV large T antigen (LT) expression downregulates TLR9 expression in epithelial and MCC-derived cells. Accordingly, silencing of LT expression results in upregulation of mRNA TLR9 levels. In addition, small T antigen (sT) also appears to inhibit TLR9 expression, since inhibition of its expression also resulted in an increase of TLR9 mRNA levels. LT inhibits TLR9 expression by decreasing the mRNA levels of the C/EBPß transactivator, a positive regulator of the TLR9 promoter. Chromatin immunoprecipitation reveals that C/EBPß binding at a C/EBPß response element (RE) in the TLR9 promoter is strongly inhibited by expression of MCPyV early genes and that mutation of the C/EBP RE prevents MCPyV downregulation of TLR9. A survey of BK polyomavirus (BKPyV), JC polyomavirus (JCPyV), KI polyomavirus (KIPyV), MCPyV, simian virus 40 (SV40), and WU polyomavirus (WUPyV) early genes revealed that only BKPyV and MCPyV are potent inhibitors of TLR9 gene expression. MCPyV LT targeting of C/EBP transactivators is likely to play an important role in viral persistence and potentially inhibit host cell immune responses during MCPyV tumorigenesis.
Assuntos
Antígenos Virais de Tumores/metabolismo , Carcinoma de Célula de Merkel/genética , Regulação para Baixo , Poliomavírus das Células de Merkel/metabolismo , Infecções por Polyomavirus/genética , Receptor Toll-Like 9/genética , Antígenos Virais de Tumores/genética , Carcinoma de Célula de Merkel/metabolismo , Carcinoma de Célula de Merkel/virologia , Interações Hospedeiro-Patógeno , Humanos , Células de Merkel/metabolismo , Células de Merkel/virologia , Poliomavírus das Células de Merkel/genética , Infecções por Polyomavirus/metabolismo , Infecções por Polyomavirus/virologia , Regiões Promotoras Genéticas , Receptor Toll-Like 9/metabolismo , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/metabolismo , Infecções Tumorais por Vírus/virologiaRESUMO
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.
Assuntos
Carcinoma de Célula de Merkel , Movimento Celular , Proliferação de Células , Poliomavírus das Células de Merkel , Proteína Fosfatase 2 , Humanos , Poliomavírus das Células de Merkel/fisiologia , Poliomavírus das Células de Merkel/genética , Proteína Fosfatase 2/metabolismo , Proteína Fosfatase 2/genética , Carcinoma de Célula de Merkel/virologia , Carcinoma de Célula de Merkel/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Cloridrato de Fingolimode/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Linhagem Celular Tumoral , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/genética , Infecções por Polyomavirus/virologia , Neoplasias Cutâneas/virologia , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 2 Dependente de Ciclina/genéticaRESUMO
Whether Merkel cells regenerate in adult skin and from which progenitor cells they regenerate are a subject of debate. Understanding Merkel cell regeneration is of interest to the study of Merkel cell carcinoma, a rare neuroendocrine skin cancer hypothesized to originate in a Merkel cell progenitor transformed by Merkel cell polyomavirus small and large T antigens. We sought to understand what the adult Merkel cell progenitors are and whether they can give rise to Merkel cell carcinoma. We used lineage tracing to identify SOX9-expressing cells (SOX9+ cells) as Merkel cell progenitors in postnatal murine skin. Merkel cell regeneration from SOX9+ progenitors occurs rarely in mature skin unless in response to minor mechanical injury. Merkel cell polyomavirus small T antigen and functional imitation of large T antigen in SOX9+ cells enforced neuroendocrine and Merkel cell lineage reprogramming in a subset of cells. These results identify SOX9+ cells as postnatal Merkel cell progenitors that can be reprogrammed by Merkel cell polyomavirus T antigens to express neuroendocrine markers.
Assuntos
Carcinoma de Célula de Merkel , Poliomavírus das Células de Merkel , Infecções por Polyomavirus , Polyomavirus , Neoplasias Cutâneas , Infecções Tumorais por Vírus , Adulto , Humanos , Camundongos , Animais , Carcinoma de Célula de Merkel/patologia , Células de Merkel , Antígenos Virais de Tumores , Neoplasias Cutâneas/patologiaRESUMO
OBJECTIVE: Cyclin-dependent kinase 1 (CDK1)/cyclin B1 phosphorylates many of the same substrates as mTORC1 (a key regulator of glucose metabolism), including the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Only mitotic CDK1 phosphorylates 4E-BP1 at residue S82 in mice (S83 in humans), in addition to the common 4E-BP1 phospho-acceptor sites phosphorylated by both CDK1 and mTORC1. We examined glucose metabolism in mice having a single aspartate phosphomimetic amino acid knock in substitution at the 4E-BP1 serine 82 (4E-BP1S82D) mimicking constitutive CDK1 phosphorylation. METHODS: Knock-in homozygous 4E-BP1S82D and 4E-BP1S82A C57Bl/6N mice were assessed for glucose tolerance testing (GTT) and metabolic cage analysis on regular and on high-fat chow diets. Gastrocnemius tissues from 4E-BP1S82D and WT mice were subject to Reverse Phase Protein Array analysis. Since the bone marrow is one of the few tissues typically having cycling cells that transit mitosis, reciprocal bone-marrow transplants were performed between male 4E-BP1S82D and WT mice, followed by metabolic assessment, to determine the role of actively cycling cells on glucose homeostasis. RESULTS: Homozygous knock-in 4E-BP1S82D mice showed glucose intolerance that was markedly accentuated with a diabetogenic high-fat diet (p = 0.004). In contrast, homozygous mice with the unphosphorylatable alanine substitution (4E-BP1S82A) had normal glucose tolerance. Protein profiling of lean muscle tissues, largely arrested in G0, did not show protein expression or signaling changes that could account for these results. Reciprocal bone-marrow transplantation between 4E-BP1S82D and wild-type littermates revealed a trend for wild-type mice with 4E-BP1S82D marrow engraftment on high-fat diets to become hyperglycemic after glucose challenge. CONCLUSIONS: 4E-BP1S82D is a single amino acid substitution that induces glucose intolerance in mice. These findings indicate that glucose metabolism may be regulated by CDK1 4E-BP1 phosphorylation independent from mTOR and point towards an unexpected role for cycling cells that transit mitosis in diabetic glucose control.
Assuntos
Proteína Quinase CDC2 , Intolerância à Glucose , Humanos , Camundongos , Masculino , Animais , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Fosfoproteínas/metabolismo , Fosforilação , Sinapsinas/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Mutação , GlucoseRESUMO
Merkel cell polyomavirus (MCV) and high-risk human papillomavirus (HPV) are human tumor viruses that cause Merkel cell carcinoma (MCC) and oropharyngeal squamous cell carcinoma (OSCC), respectively. HPV E7 and MCV large T (LT) oncoproteins target the retinoblastoma tumor suppressor protein (pRb) through the conserved LxCxE motif. We identified enhancer of zeste homolog 2 (EZH2) as a common host oncoprotein activated by both viral oncoproteins through the pRb binding motif. EZH2 is a catalytic subunit of the polycomb 2 (PRC2) complex that trimethylates histone H3 at lysine 27 (H3K27me3). In MCC tissues EZH2 was highly expressed, irrespective of MCV status. Loss-of-function studies revealed that viral HPV E6/E7 and T antigen expression are required for Ezh2 mRNA expression and that EZH2 is essential for HPV(+)OSCC and MCV(+)MCC cell growth. Furthermore, EZH2 protein degraders reduced cell viability efficiently and rapidly in HPV(+)OSCC and MCV(+)MCC cells, whereas EZH2 histone methyltransferase inhibitors did not affect cell proliferation or viability within the same treatment period. These results suggest that a methyltransferase-independent function of EZH2 contributes to tumorigenesis downstream of two viral oncoproteins, and that direct targeting of EZH2 protein expression could be a promising strategy for the inhibition of tumor growth in HPV(+)OSCC and MCV(+)MCC patients.
Assuntos
Carcinoma de Célula de Merkel , Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Polyomavirus , Neoplasias Cutâneas , Humanos , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Papillomavirus Humano , Infecções por Papillomavirus/complicações , Metiltransferases , Carcinoma de Célula de Merkel/metabolismo , Proteínas Oncogênicas Virais/genética , Neoplasias Cutâneas/metabolismoRESUMO
4E-BP1 is a tumor suppressor regulating cap-dependent translation that is in turn controlled by mechanistic target of rapamycin (mTOR) or cyclin-dependent kinase 1 (CDK1) phosphorylation. 4E-BP1 serine 82 (S82) is phosphorylated by CDK1, but not mTOR, and the consequences of this mitosis-specific phosphorylation are unknown. Knock-in mice were generated with a single 4E-BP1 S82 alanine (S82A) substitution leaving other phosphorylation sites intact. S82A mice were fertile and exhibited no gross developmental or behavioral abnormalities, but the homozygotes developed diffuse and severe polycystic liver and kidney disease with aging, and lymphoid malignancies after irradiation. Sublethal irradiation caused immature T-cell lymphoma only in S82A mice while S82A homozygous mice have normal T-cell hematopoiesis before irradiation. Whole genome sequencing identified PTEN mutations in S82A lymphoma and impaired PTEN expression was verified in S82A lymphomas derived cell lines. Our study suggests that the absence of 4E-BP1S82 phosphorylation, a subtle change in 4E-BP1 phosphorylation, might predispose to polycystic proliferative disease and lymphoma under certain stressful circumstances, such as aging and irradiation.
Assuntos
Proteína Quinase CDC2 , Linfoma , Camundongos , Animais , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Fosforilação , Serina/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linfoma/genéticaRESUMO
Merkel cell polyomavirus (MCV) causes one of the most aggressive human skin cancers, but laboratory studies on MCV replication have proven technically difficult. We report the first recombinase-mediated MCV minicircle (MCVmc) system that generates high levels of circularized virus, allowing facile MCV genetic manipulation and characterization of viral gene expression kinetics during replication. Mutations to Fbw7, Skp2, ß-TrCP and hVam6p interaction sites, or to the stem loop sequence for the MCV-encoded miRNA precursor, markedly increase viral replication, whereas point mutation to an origin-binding site eliminates active virus replication. To further increase the utility of this system, an mScarlet fusion protein was inserted into the VP1 c-terminus to generate a non-infectious reporter virus for studies on virus kinetics. When this reporter virus genome is heterologously expressed together with MCV VP1 and VP2, virus-like particles are generated. The reporter virus genome is encapsidated and can be used at lower biosafety levels for one-round infection studies. Our findings reveal that MCV has multiple, self-encoded viral restriction mechanisms to promote viral latency over lytic replication, and these mechanisms are now amenable to examination using a recombinase technology.
Assuntos
Poliomavírus das Células de Merkel , Infecções por Polyomavirus , Polyomavirus , Infecções Tumorais por Vírus , Antígenos Virais de Tumores/genética , Humanos , Cinética , Poliomavírus das Células de Merkel/genética , Poliomavírus das Células de Merkel/metabolismo , Polyomavirus/genética , Polyomavirus/metabolismo , Recombinases/metabolismo , Replicação Viral/genéticaRESUMO
Although KIT-mutant GISTs can be effectively treated with tyrosine kinase inhibitors (TKIs), many patients develop resistance to imatinib mesylate (IM) as well as the FDA-approved later-line agents sunitinib, regorafenib and ripretinib. Resistance mechanisms mainly involve secondary mutations in the KIT receptor tyrosine kinase gene indicating continued dependency on the KIT signaling pathway. The fact that the type of secondary mutation confers either sensitivity or resistance towards TKIs and the notion that secondary mutations exhibit intra- and intertumoral heterogeneity complicates the optimal choice of treatment in the imatinib-resistant setting. Therefore, new strategies that target KIT independently of its underlying mutations are urgently needed. Homoharringtonine (HHT) is a first-in-class inhibitor of protein biosynthesis and is FDA-approved for the treatment of chronic myeloid leukemia (CML) that is resistant to at least two TKIs. HHT has also shown activity in KIT-mutant mastocytosis models, which are intrinsically resistant to imatinib and most other TKIs. We hypothesized that HHT could be effective in GIST through downregulation of KIT expression and subsequent decrease of KIT activation and downstream signaling. Testing several GIST cell line models, HHT led to a significant reduction in nascent protein synthesis and was highly effective in the nanomolar range in IM-sensitive and IM-resistant GIST cell lines. HHT treatment resulted in a rapid and complete abolishment of KIT expression and activation, while KIT mRNA levels were minimally affected. The response to HHT involved induction of apoptosis as well as cell cycle arrest. The antitumor activity of HHT was confirmed in a GIST xenograft model. Taken together, inhibition of protein biosynthesis is a promising strategy to overcome TKI resistance in GIST.
Assuntos
Antineoplásicos , Neoplasias Gastrointestinais , Tumores do Estroma Gastrointestinal , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/patologia , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Mepesuccinato de Omacetaxina/farmacologia , Humanos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/metabolismoRESUMO
Merkel cell carcinoma (MCC) is the most aggressive skin cancer. Recently, it was demonstrated that human Merkel cell polyomavirus (MCV) is clonally integrated in approximately 80% of MCC tumors. However, direct evidence for whether oncogenic viral proteins are needed for the maintenance of MCC cells is still missing. To address this question, we knocked down MCV T-antigen (TA) expression in MCV-positive MCC cell lines using three different short hairpin RNA (shRNA)-expressing vectors targeting exon 1 of the TAs. The MCC cell lines used include three newly generated MCV-infected cell lines and one MCV-negative cell line from MCC tumors. Notably, all MCV-positive MCC cell lines underwent growth arrest and/or cell death upon TA knockdown, whereas the proliferation of MCV-negative cell lines remained unaffected. Despite an increase in the number of annexin V-positive, 7-amino-actinomycin D (7-AAD)-negative cells upon TA knockdown, activation of caspases or changes in the expression and phosphorylation of Bcl-2 family members were not consistently detected after TA suppression. Our study provides the first direct experimental evidence that TA expression is necessary for the maintenance of MCV-positive MCC and that MCV is the infectious cause of MCV-positive MCC.