RESUMEN
Intracellular pathogens have evolved to utilize normal cellular processes to complete their replicative cycles. Pathogens that interface with proliferative cell signaling pathways risk infections that can lead to cancers, but the factors that influence malignant outcomes are incompletely understood. Human papillomaviruses (HPVs) predominantly cause benign hyperplasia in stratifying epithelial tissues. However, a subset of carcinogenic or "high-risk" HPV (hr-HPV) genotypes are etiologically linked to nearly 5% of all human cancers. Progression of hr-HPV-induced lesions to malignancies is characterized by increased expression of the E6 and E7 oncogenes and the oncogenic functions of these viral proteins have been widely studied. Yet, the mechanisms that regulate hr-HPV oncogene transcription and suppress their expression in benign lesions remain poorly understood. Here, we demonstrate that EGFR/MEK/ERK signaling, influenced by epithelial contact inhibition and tissue differentiation cues, regulates hr-HPV oncogene expression. Using monolayer cells, epithelial organotypic tissue models, and neoplastic tissue biopsy materials, we show that cell-extrinsic activation of ERK overrides cellular control to promote HPV oncogene expression and the neoplastic phenotype. Our data suggest that HPVs are adapted to use the EGFR/MEK/ERK signaling pathway to regulate their productive replicative cycles. Mechanistic studies show that EGFR/MEK/ERK signaling influences AP-1 transcription factor activity and AP-1 factor knockdown reduces oncogene transcription. Furthermore, pharmacological inhibitors of EGFR, MEK, and ERK signaling quash HPV oncogene expression and the neoplastic phenotype, revealing a potential clinical strategy to suppress uncontrolled cell proliferation, reduce oncogene expression and treat HPV neoplasia.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/complicaciones , Neoplasias del Cuello Uterino/virología , Quinasas MAP Reguladas por Señal Extracelular/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Terapia Molecular Dirigida , Proteínas Oncogénicas Virales/genética , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/virología , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/terapiaRESUMEN
Human papillomavirus (HPV) infections are transmitted through sexual or other close contact and are etiologically associated with epithelial warts, papillomas, and intraepithelial lesions that may progress to cancer. Indeed, 4.8% of the global cancer burden is linked to HPV infection. Highly effective vaccines protect against two to nine of the most medically important HPV genotypes, yet vaccine uptake is inadequate and/or cost prohibitive in many settings. With HPV-related cancer incidence expected to rise over the coming decades, there is a need for effective HPV microbicides. Herein, we demonstrate the strong inhibitory activity of the heparin-neutralizing drug protamine sulfate (PS) against HPV infection. Pretreatment of cells with PS greatly reduced infection, regardless of HPV genotype or virus source. Vaginal application of PS prevented infection of the murine genital tract by HPV pseudovirions. Time-of-addition assays where PS was added to cells before infection, during infection, or after viral attachment demonstrated strong inhibitory activities on early infection steps. No effect on virus infection was found for cell lines deficient in heparan sulfate expression, suggesting that PS binds to heparan sulfate on the cell surface. Consistent with this, prophylactic PS exposure prevented viral attachment, including under low-pH conditions akin to the human vaginal tract. Our findings suggest PS acts dually to prevent HPV infection: prophylactic treatment prevents HPV attachment to host cells, and postattachment administration alters viral entry. Clinical trials are warranted to determine whether protamine-based products are effective as topical microbicides against genital HPVs.
Asunto(s)
Infecciones por Papillomavirus , Animales , Línea Celular , Femenino , Humanos , Ratones , Papillomaviridae , Infecciones por Papillomavirus/tratamiento farmacológico , Infecciones por Papillomavirus/epidemiología , Infecciones por Papillomavirus/prevención & control , Protaminas/farmacología , Internalización del VirusRESUMEN
The mammalian target of rapamycin (mTOR) downstream of phosphatidylinositol 3-kinase (PI3K) in the growth factor receptor (GFR) pathway is a crucial metabolic sensor that integrates growth factor signals in cells. We recently showed that human papillomavirus (HPV) type 16 exposure activates signaling from GFRs in human keratinocytes. Thus, we predicted that the virus would induce the PI3K/mTOR pathway upon interaction with host cells. We detected activation of Akt and mTOR several minutes following exposure of human keratinocytes to HPV type 16 (HPV16) pseudovirions. Activated mTOR induced phosphorylation of the mTOR complex 1 substrates 4E-BP1 and S6K, which led to induction of the functional protein translational machinery. Blockade of epidermal GFR (EGFR) signaling revealed that each of these events is at least partially dependent upon EGFR activation. Importantly, activation of PI3K/Akt/mTOR signaling inhibited autophagy in the early stages of virus-host cell interaction. Biochemical and genetic approaches revealed critical roles for mTOR activation and autophagy suppression in HPV16 early infection events. In summary, the HPV-host cell interaction stimulates the PI3K/Akt/mTOR pathway and inhibits autophagy, and in combination these events benefit virus infection.
Asunto(s)
Autofagia , Papillomavirus Humano 16/fisiología , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Internalización del Virus , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular , Línea Celular , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Células HEK293 , Humanos , Queratinocitos/metabolismo , Queratinocitos/virología , Fosfohidrolasa PTEN/metabolismo , Infecciones por Papillomavirus/metabolismo , Fosfatidilinositol 3-Quinasa/genética , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/genética , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Human papillomaviruses (HPVs) are a significant public health concern due to their widespread transmission, morbidity, and oncogenic potential. Despite efficacious vaccines, millions of unvaccinated individuals and those with existing infections will develop HPV-related diseases for the next two decades and beyond. The continuing burden of HPV-related diseases is exacerbated by the lack of effective therapies or cures for infections, highlighting the need to identify and develop antivirals. The experimental murine papillomavirus type 1 (MmuPV1) model provides opportunities to study papillomavirus pathogenesis in cutaneous epithelium, the oral cavity, and the anogenital tract. However, to date the MmuPV1 infection model has not been used to demonstrate the effectiveness of potential antivirals. We previously reported that inhibitors of cellular MEK/ERK signaling suppress oncogenic HPV early gene expression in three-dimensional tissue cultures. Herein, we adapted the MmuPV1 infection model to determine whether MEK inhibitors have anti-papillomavirus properties in vivo. We demonstrate that oral delivery of a MEK1/2 inhibitor promotes papilloma regression in immunodeficient mice that otherwise would have developed persistent infections. Quantitative histological analyses reveal that inhibition of MEK/ERK signaling reduces E6/E7 mRNA, MmuPV1 DNA, and L1 protein expression within MmuPV1-induced lesions. These data suggest that MEK1/2 signaling is essential for both early and late MmuPV1 replication events supporting our previous findings with oncogenic HPVs. We also provide evidence that MEK inhibitors protect mice from developing secondary tumors. Thus, our data suggest that MEK inhibitors have potent antiviral and anti-tumor properties in a preclinical mouse model and merit further investigation as papillomavirus antiviral therapies.
Asunto(s)
Neoplasias , Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Humanos , Animales , Ratones , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/tratamiento farmacológico , Virus del Papiloma Humano , Carcinogénesis , Quinasas de Proteína Quinasa Activadas por Mitógenos , Papillomaviridae/genética , Proteínas Oncogénicas Virales/metabolismoRESUMEN
Human papillomaviruses (HPVs) are a significant public health concern due to their widespread transmission, morbidity, and oncogenic potential. Despite efficacious vaccines, millions of unvaccinated individuals and those with existing infections will develop HPV-related diseases for the next two decades. The continuing burden of HPV-related diseases is exacerbated by the lack of effective therapies or cures for most infections, highlighting the need to identify and develop antivirals. The experimental murine papillomavirus type 1 (MmuPV1) model provides opportunities to study papillomavirus pathogenesis in cutaneous epithelium, the oral cavity, and the anogenital tract. However, to date the MmuPV1 infection model has not been used to demonstrate the effectiveness of potential antivirals. We previously reported that inhibitors of cellular MEK/ERK signaling suppress oncogenic HPV early gene expression in vitro . Herein, we adapted the MmuPV1 infection model to determine whether MEK inhibitors have anti-papillomavirus properties in vivo . We demonstrate that oral delivery of a MEK1/2 inhibitor promotes papilloma regression in immunodeficient mice that otherwise would have developed persistent infections. Quantitative histological analyses revealed that inhibition of MEK/ERK signaling reduces E6/E7 mRNAs, MmuPV1 DNA, and L1 protein expression within MmuPV1-induced lesions. These data suggest that MEK1/2 signaling is essential for both early and late MmuPV1 replication events supporting our previous findings with oncogenic HPVs. We also provide evidence that MEK inhibitors protect mice from developing secondary tumors. Thus, our data suggest that MEK inhibitors have potent anti-viral and anti-tumor properties in a preclinical mouse model and merit further investigation as papillomavirus antiviral therapies. Significance Statement: Persistent human papillomavirus (HPV) infections cause significant morbidity and oncogenic HPV infections can progress to anogenital and oropharyngeal cancers. Despite the availability of effective prophylactic HPV vaccines, millions of unvaccinated individuals, and those currently infected will develop HPV-related diseases over the next two decades and beyond. Thus, it remains critical to identify effective antivirals against papillomaviruses. Using a mouse papillomavirus model of HPV infection, this study reveals that cellular MEK1/2 signaling supports viral tumorigenesis. The MEK1/2 inhibitor, trametinib, demonstrates potent antiviral activities and promotes tumor regression. This work provides insight into the conserved regulation of papillomavirus gene expression by MEK1/2 signaling and reveals this cellular pathway as a promising therapeutic target for the treatment of papillomavirus diseases.
RESUMEN
BACKGROUND: Recent publications from a single research group have suggested that aldehyde-based high-level disinfectants (HLDs), such as ortho-phthalaldehyde (OPA), are not effective at inactivating HPVs and that therefore, patients may be at risk of HPV infection from medical devices. These results could have significant public health consequences and therefore necessitated evaluation of their reproducibility and clinical relevance. METHODS: We developed methods and used standardised controls to: (1) quantify the infectious levels of clinically-sourced HPVs from patient lesions and compare them to laboratory-derived HPVs, (2) evaluate experimental factors that should be controlled to ensure consistent and reproducible infectivity measurements of different HPV genotypes, and (3) determine the efficacy of select HLDs. FINDINGS: A novel focus forming unit (FFU) infectivity assay demonstrated that exfoliates from patient anogenital lesions and respiratory papillomas yielded infectious HPV burdens up to 2.7 × 103 FFU; therefore, using 2.2 × 102 to 1.0 × 104 FFU of laboratory-derived HPVs in disinfection assays provides a relevant range for clinical exposures. RNase and neutralising antibody sensitivities were used to ensure valid infectivity measures of tissue-derived and recombinant HPV preparations. HPV infectivity was demonstrated over a dynamic range of 4-5 log10; and disinfection with OPA and hypochlorite was achieved over 3 to >4 log10 with multiple genotypes of tissue-derived and recombinant HPV isolates. INTERPRETATION: This work, along with a companion publication from an independent lab in this issue, address a major public health question by showing that HPVs are susceptible to HLDs. FUNDING: Advanced Sterilization Products; US NIH (R01CA207368, U19AI084081, P30CA118100).
Asunto(s)
Alphapapillomavirus/efectos de los fármacos , Alphapapillomavirus/fisiología , Desinfectantes/farmacología , Infecciones por Papillomavirus/virología , Carga Viral , Alphapapillomavirus/clasificación , Alphapapillomavirus/genética , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Línea Celular , Células Cultivadas , Desinfección/métodos , Femenino , Genoma Viral , Genotipo , Humanos , Masculino , Pruebas de NeutralizaciónRESUMEN
Chromatin remodeling is emerging as a critical regulator of DNA repair factor access to DNA damage, and optimum accessibility of these factors is a major determinant of DNA repair outcome. Hence, chromatin remodeling is likely to play a key role in genome stabilization and tumor suppression. We previously showed that nucleosome eviction near double-strand breaks (DSBs) in yeast is regulated by the INO80 nucleosome remodeling complex and is defective in mutants lacking the Arp8 subunit of INO80. In the absence of homologous donor sequences, RPA recruitment to a DSB appeared normal in arp8Delta, but Rad51 recruitment was defective. We now show that the early strand invasion step of homologous recombination (HR) is markedly delayed in an arp8Delta haploid, but there is only a minor defect in haploid HR efficiency (MAT switching). In an arp8Delta diploid, interhomolog DSB repair by HR shows a modest defect that is partially suppressed by overexpression of Rad51 or its mediator, Rad52. In wild type cells, DSB repair typically results in gene conversion, and most gene conversion tracts are continuous, reflecting efficient mismatch repair of heteroduplex DNA. In contrast, arp8Delta gene conversion tracts are longer and frequently discontinuous, indicating defects in late stages of HR. Interestingly, when a homologous donor sequence is present, Rad51 is recruited normally to a DSB in arp8Delta, but its transfer to the donor is delayed, and this correlates with defective displacement of donor nucleosomes. We propose that retained nucleosomes at donors destabilize heteroduplex DNA or impair mismatch recognition, reflected in delayed strand invasion and altered conversion tracts.
Asunto(s)
Ensamble y Desensamble de Cromatina , Conversión Génica , Mitosis/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN/fisiología , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas de Microfilamentos/deficiencia , Nucleosomas/metabolismo , Recombinasa Rad51/biosíntesis , Proteína Recombinante y Reparadora de ADN Rad52/biosíntesis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Homología de Secuencia de Ácido Nucleico , Regulación hacia ArribaRESUMEN
RecQ helicases maintain genome stability and suppress tumors in higher eukaryotes through roles in replication and DNA repair. The yeast RecQ homolog Sgs1 interacts with Top3 topoisomerase and Rmi1. In vitro, Sgs1 binds to and branch migrates Holliday junctions (HJs) and the human RecQ homolog BLM, with Top3alpha, resolves synthetic double HJs in a noncrossover sense. Sgs1 suppresses crossovers during the homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Crossovers are associated with long gene conversion tracts, suggesting a model in which Sgs1 helicase catalyzes reverse branch migration and convergence of double HJs for noncrossover resolution by Top3. Consistent with this model, we show that allelic crossovers and gene conversion tract lengths are increased in sgs1Delta. However, crossover and tract length suppression was independent of Sgs1 helicase activity, which argues against helicase-dependent HJ convergence. HJs may converge passively by a "random walk," and Sgs1 may play a structural role in stimulating Top3-dependent resolution. In addition to the new helicase-independent functions for Sgs1 in crossover and tract length control, we define three new helicase-dependent functions, including the suppression of chromosome loss, chromosome missegregation, and synthetic lethality in srs2Delta. We propose that Sgs1 has helicase-dependent functions in replication and helicase-independent functions in DSB repair by HR.
Asunto(s)
Intercambio Genético/genética , ADN Helicasas/metabolismo , Conversión Génica/genética , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/metabolismo , Segregación Cromosómica/efectos de los fármacos , Cromosomas Fúngicos/genética , Daño del ADN/genética , ADN Helicasas/genética , Metilmetanosulfonato/farmacología , Mutación/genética , RecQ Helicasas , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The yeast Mre11-Rad50-Xrs2 (MRX) and Ku complexes regulate single-strand resection at DNA double-strand breaks (DSB), a key early step in homologous recombination (HR). A prior plasmid gap repair study showed that mre11 mutations, which slow single-strand resection, reduce gene conversion tract lengths and the frequency of associated crossovers. Here we tested whether mre11Delta or nuclease-defective mre11 mutations reduced gene conversion tract lengths during HR between homologous chromosomes in diploid yeast. We found that mre11 mutations reduced the efficiency of HR but did not reduce tract lengths or crossovers, despite substantially reduced end-resection at the test (ura3) locus. End-resection is increased in yku70Delta, but this change also had no effect on tract lengths. Thus, heteroduplex formation and tract lengths are not regulated by the extent of end-resection during DSB repair in a chromosomal context. In a plasmid-chromosome DSB repair assay, tract lengths were again similar in wild-type and mre11Delta, but they were reduced in mre11Delta in a gap repair assay. These results indicate that tract lengths are not affected by the extent of end processing when broken ends can invade nearby sites, perhaps because MRX coordination of the two broken ends is dispensable when ends invade nearby sites. Although HR outcome was largely unaffected in mre11 mutants, break-induced replication (BIR) and chromosome loss increased, suggesting that Mre11 function in mitotic HR is limited to early HR stages. Interestingly, yku70Delta suppressed BIR in mre11 mutants. BIR is also elevated in rad51 mutants, but yku70Delta did not suppress BIR in a rad51 background. These results indicate that Mre11 functions in Rad51-independent BIR, and that Ku functions in Rad51-dependent BIR.
Asunto(s)
Antígenos Nucleares/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/fisiología , Endodesoxirribonucleasas/fisiología , Exodesoxirribonucleasas/fisiología , Conversión Génica , Proteínas de Saccharomyces cerevisiae/fisiología , Alelos , Mapeo Cromosómico , Cromosomas Fúngicos , Daño del ADN , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Genotipo , Autoantígeno Ku , Mutación , Plásmidos/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
Chromosomal translocations are common in leukemia, but little is known about their mechanism. Metnase (also termed SETMAR) is a fusion of a histone methylase and transposase protein that arose specifically in primates. Transposases were thought to be extinct in primates because they would mediate deleterious DNA movement. In primates, Metnase interacts with DNA Ligase IV (Lig IV) and promotes nonhomologous end-joining (NHEJ) DNA repair. We show here that the primate-specific protein Metnase can also enhance NHEJ in murine cells and can also interact with murine Lig IV, indicating that it integrated into the preexisting NHEJ pathway after its development in primates. Significantly, expressing Metnase in murine cells significantly reduces chromosomal translocations. We propose that the fusion of the histone methylase SET domain and the transposase domain in the anthropoid lineage to form primate Metnase promotes accurate intrachromosomal NHEJ and thereby suppresses interchromosomal translocations. Metnase may have been selected for because it has a function opposing transposases and may thus play a key role in suppressing translocations that underlie oncogenicity.