RESUMEN
Integration, which leads to the disruption of the circular HPV genome, is considered as a critical, albeit not obligatory, step in carcinogenic progression. Although cervical carcinomas with extrachromosomal HPV plasmid genomes have been described, the virus is integrated in 70% of HPV16-positive cervical tumours. Limited information is available about HPV integration in head and neck tumours (HNC). In this study, we have characterised the physical status of HPV in a set of tonsillar tumour samples using different methods--the mapping of E2 integration breakpoint at the mRNA level, the 3' RACE based Amplification of Papillomavirus Oncogene Transcripts (APOT) assay and Southern blot. Furthermore, the impact of HPV integration on patients' prognosis has been evaluated in a larger set of 186 patients with head and neck cancer. Based on the analysis of E2 mRNA, HPV was integrated in the host genome in 43% of the HPV-positive samples. Extrachromosomal or mixed form was present in 57%. In fresh frozen samples, the APOT and E2 mapping results were in agreement. The results were confirmed using Southern blotting. Furthermore, the type and exact site of integration were determined. The survival analysis of 186 patients revealed HPV positivity, tumour size and lymph node positivity as factors that influence disease specific survival. However, no statistically significant difference was found in disease specific survival between patients with HPV-positive integrated vs. extrachromosomal/mixed forms of the virus.
Asunto(s)
Carcinoma de Células Escamosas/virología , Neoplasias de Cabeza y Cuello/virología , Infecciones por Papillomavirus/virología , Neoplasias Tonsilares/virología , Integración Viral/genética , Southern Blotting , Femenino , Neoplasias de Cabeza y Cuello/mortalidad , Humanos , Masculino , Infecciones por Papillomavirus/complicaciones , Pronóstico , Modelos de Riesgos Proporcionales , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
BACKGROUND: Better insights into the molecular changes involved in virus-associated and -independent head and neck cancer may advance our knowledge of HNC carcinogenesis and identify critical disease biomarkers. Here we aimed to characterize the expression profiles in a matched set of well-characterized HPV-dependent and HPV-independent tonsillar tumors and equivalent immortalized keratinocyte clones to define potential and clinically relevant biomarkers of HNC of different etiology. METHODS: Fresh frozen tonsillar cancer tissues were analyzed together with non-malignant tonsillar tissues and compared with cervical tumors and normal cervical tissues. Furthermore, relative miRNAs abundance levels of primary and immortalized human keratinocyte clones were evaluated. The global quantitation of miRNA gene abundance was performed using a TaqMan Low Density Array system. The confirmation of differentially expressed miRNAs was performed on a set of formalin-fixed paraffin-embedded tumor samples enriched for the tumor cell fraction by macrodissection. RESULTS: We defined 46 upregulated and 31 downregulated miRNAs characteristic for the HPV-positive tonsillar tumors and 42 upregulated miRNAs and 42 downregulated miRNAs characteristic for HPV-independent tumors. In comparison with the expression profiles in cervical tumors, we defined miR-141-3p, miR-15b-5p, miR-200a-3p, miR-302c-3p, and miR-9-5p as specific for HPV induced malignancies. MiR-335-5p, miR-579-3p, and miR-126-5p were shared by the expression profiles of HPV-positive tonsillar tumors and of the HPV immortalized keratinocyte clones, whereas miR-328-3p, miR-34c-3p, and miR-885-5p were shared by the miRNA profiles of HPV-negative tonsillar tumors and the HPV-negative keratinocytes. CONCLUSIONS: We identified the miRNAs characteristic for HPV-induced tumors and tonsillar tumors of different etiology, and the results were compared with those of the model system. Our report presents the basis for further investigations leading to the identification of clinically relevant diagnostic and/or therapeutic biomarkers for tumors of viral and non-viral etiology.
Asunto(s)
Queratinocitos/citología , MicroARNs/genética , Infecciones por Papillomavirus/genética , Neoplasias Tonsilares/genética , Neoplasias del Cuello Uterino/genética , Células Cultivadas , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Queratinocitos/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Componente Principal , Neoplasias Tonsilares/virología , Neoplasias del Cuello Uterino/virologíaRESUMEN
Interferons (IFNs) have been used to treat epithelial lesions caused by human papillomavirus (HPV) persistence. Here, we exposed primary human keratinocytes (HFKs) immortalized by persistently replicating HPV-16 plasmid genomes to increasing levels of IFN-γ. While untreated HFKs retained replicating HPV-16 plasmids for up to 60-120 population doublings, IFN led to rapid HPV-16 plasmid loss. However, treated cultures eventually gave rise to outgrowth of clones harboring integrated HPV-16 genomes expressing viral E6 and E7 oncogenes from chimeric virus-cell mRNAs similar to those in cervical and head and neck cancers. Surprisingly, every HPV-16 integrant that arose after IFN exposure stemmed from an independent integration event into a different cellular gene locus, even within parallel cultures started from small cell inocula and cultured separately for ≥ 25 doublings to permit the rise and expansion of spontaneous integrants. While IFN treatment conferred a growth advantage upon preexisting integrants added to mixed control cultures, our results indicate that IFN exposure directly or indirectly induces HPV-16 integration, rather than only selects preexisting, spontaneous integrants that appear to be much less frequent. We estimate that IFN exposure increased integration rates by ≥ 100-fold. IFN-induced HPV-16 integration involved a wide range of chromosomal loci with less apparent selection for recurrent insertions near genes involved in cancer-related pathways. We conclude that IFNs and other potential treatments targeting high-risk HPV persistence that disrupt viral genome replication may promote increased high-risk HPV integration as a step in cancer progression. Therapies against high-risk HPV persistence thus need to be evaluated for their integration-inducing potential.
Asunto(s)
Herencia Extracromosómica , Genoma Viral/efectos de los fármacos , Papillomavirus Humano 16/genética , Interferón gamma/farmacología , Queratinocitos/efectos de los fármacos , Infecciones por Papillomavirus/genética , Plásmidos/genética , Integración Viral/genética , Antivirales/farmacología , Transformación Celular Viral/efectos de los fármacos , Células Cultivadas , ADN Viral/genética , Humanos , Queratinocitos/virología , Infecciones por Papillomavirus/virología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Many human papillomavirus (HPV)-positive high-grade lesions and cancers of the uterine cervix harbor integrated HPV genomes expressing the E6 and E7 oncogenes from chimeric virus-cell mRNAs, but less is known about HPV integration in head and neck cancer (HNC). Here we compared viral DNA status and E6-E7 mRNA sequences in HPV-16-positive HNC tumors to those in independent human keratinocyte cell clones derived from primary tonsillar or foreskin epithelia immortalized with HPV-16 genomes. Three of nine HNC tumors and epithelial clones containing unintegrated HPV-16 genomes expressed mRNAs spliced from HPV-16 SD880 to SA3358 and terminating at the viral early gene p(A) signal. In contrast, most integrated HPV genomes in six HNCs and a set of 31 keratinocyte clones expressed HPV-16 major early promoter (MEP)-initiated mRNAs spliced from viral SD880 directly to diverse cellular sequences, with a minority spliced to SA3358 followed by a cellular DNA junction. Sequence analysis of chimeric virus-cell mRNAs from HNC tumors and keratinocyte clones identified viral integration sites in a variety of chromosomes, with some located in or near growth control genes, including the c-myc protooncogene and the gene encoding FAP-1 phosphatase. Taken together, these findings support the hypothesis that HPV integration in cancers is a stochastic process resulting in clonal selection of aggressively expanding cells with altered gene expression of integrated HPV genomes and potential perturbations of cellular genes at or near viral integration sites. Furthermore, our results demonstrate that this selection also takes place and can be studied in primary human keratinocytes in culture.
Asunto(s)
Neoplasias de Cabeza y Cuello/virología , Papillomavirus Humano 16/genética , Queratinocitos/virología , ARN Mensajero/metabolismo , Recombinación Genética , Integración Viral , Transformación Celular Viral , Células Cultivadas , Células Clonales/virología , Femenino , Genoma Viral , Papillomavirus Humano 16/metabolismo , Humanos , Queratinocitos/metabolismo , Masculino , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , ARN Mensajero/genética , ARN Viral/genética , ARN Viral/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Neoplasias del Cuello Uterino/virologíaRESUMEN
High-risk human papillomavirus types (HPV-HR) are associated with head and neck cancer (HNC) risk and better survival. Most patients with HPV-HR DNA-positive tumors develop anti-HPV E6/E7 antibodies; however, it is unclear whether those who mount an immune response have similar risk factors or clinical outcomes as those who do not. HPV-16 DNA tumor-positive HNC cases were evaluated for HPV-16 E6 and E7 antibodies using a GST capture ELISA system. Among 57 HPV-16 DNA tumor-positive HNC cases, 67% were detected with HPV-16 E6 and/or E7 antibodies. Male gender (76% vs. 42%, p = 0.02), younger age (63% vs. 16%, p = 0.001) but not tobacco or alcohol were associated with E6 and/or E7 seropositivity. Seropositivity was associated more often with late stage (76%), poor grade (65%), positive nodes (82%). and in the oropharynx (82%), Median disease-specific and recurrence-free survival were longer in E6 and/or E7 seropositive compared to E6/E7-negative cases (2.2 years vs. 1.4 years, both outcomes), although results were not statistically significant. When examined jointly with p16 expression, E6 and/or E7-positive/p16-positive cases had better disease-specific (2.1 years vs. 1.1 years, p = 0.06) and recurrence-free (2.3 years vs. 1.1 years, p = 0.03) survival compared to E6-/E7-/p16- cases. These findings suggest there are 2 distinct HNC patient groups with HPV DNA-positive tumors, distinguishable by E6 and/or E7 antibody status. Differences in antibody status are associated with distinct risk factors and clinical outcomes. This information can be available as a simple blood test at initial presentation, before the removal of tissue through biopsy or surgery.
Asunto(s)
Anticuerpos Antivirales/análisis , Neoplasias de Cabeza y Cuello/virología , Proteínas Oncogénicas Virales/inmunología , Papillomaviridae/inmunología , Proteínas E7 de Papillomavirus/inmunología , Proteínas Represoras/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Papillomaviridae/aislamiento & purificación , Factores de Riesgo , Tasa de SupervivenciaRESUMEN
Human papillomavirus (HPV) DNAs isolated from cervical and head and neck carcinomas frequently contain nucleotide sequence alterations in the viral upstream regulatory region (URR). Our study has addressed the role such sequence changes may play in the efficiency of establishing HPV persistence and altered keratinocyte growth. Genomic mapping of integrated HPV type 16 (HPV-16) genomes from 32 cervical cancers revealed that the viral E6 and E7 oncogenes, as well as the L1 region/URR, were intact in all of them. The URR sequences from integrated and unintegrated viral DNA were found to harbor distinct sets of nucleotide substitutions. A subset of the altered URRs increased the potential of HPV-16 to establish persistent, cell growth-altering viral-genome replication in the cell. This aggressive phenotype in culture was not solely due to increased viral early gene transcription, but also to augmented initial amplification of the viral genome. As revealed in a novel ori-dependent HPV-16 plasmid amplification assay, the altered motifs that led to increased viral transcription from the intact genome also greatly augmented HPV-16 ori function. The nucleotide sequence changes correlate with those previously described in the distinct geographical North American type 1 and Asian-American variants that are associated with more aggressive disease in epidemiologic studies and encompass, but are not limited to, alterations in previously characterized sites for the negative regulatory protein YY1. Our results thus provide evidence that nucleotide alterations in HPV regulatory sequences could serve as potential prognostic markers of HPV-associated carcinogenesis.
Asunto(s)
Carcinoma/virología , Transformación Celular Viral , Papillomavirus Humano 16/genética , Infecciones por Papillomavirus/virología , Secuencias Reguladoras de Ácidos Nucleicos , Origen de Réplica , Transcripción Genética , Neoplasias del Cuello Uterino/virología , Secuencia de Bases , Línea Celular , Femenino , Papillomavirus Humano 16/aislamiento & purificación , Papillomavirus Humano 16/fisiología , Humanos , Queratinocitos/virología , Datos de Secuencia MolecularRESUMEN
Mucosal high-risk (HR) human papillomaviruses (HPVs) that cause cervical and other anogenital cancers also are found in approximately 25% of head and neck carcinomas (HNCs), especially those arising in the oropharynx and the tonsils. While many HR HPV types are common in anogenital cancer, over 90% of HPV-positive HNCs harbor HPV type 16 (HPV-16). Using a quantitative colony-forming assay, we compared the ability of full-length mucosal HPV genomes, i.e., the low-risk HPV-11 and HR HPV-16, -18, and -31, to persist in and alter the growth of primary human keratinocytes from the foreskin, cervix, and tonsils. The HR HPV types led to the formation of growing keratinocyte colonies in culture independent of the site of epithelial origin. However, HPV-18 induced colony growth in all keratinocytes >4-fold more effectively than HPV-16 or HPV-31 and >20-fold more efficiently than HPV-11 or controls. HPV-11-transfected or control colonies failed to expand beyond 32 to 36 population doublings postexplantation. In contrast, individual HR HPV-transfected clones exhibited no apparent slowdown of growth or "crisis," and many maintained HPV plasmid persistence beyond 60 population doublings. Keratinocyte clones harboring extrachromosomal HR HPV genomes had shorter population doubling times and formed dysplastic stratified epithelia in organotypic raft cultures, mirroring the pathological features of higher-grade intraepithelial lesions, yet did not exhibit chromosomal instability. We conclude that, in culture, the HR HPV type, rather than the site of epithelial origin of the cells, determines the efficacy of inducing continued growth of individual keratinocytes, with HPV-18 being the most aggressive mucosal HR HPV type tested.
Asunto(s)
Papillomavirus Humano 18/fisiología , Queratinocitos/virología , Southern Blotting , Células Cultivadas , Cuello del Útero/citología , Cuello del Útero/virología , Femenino , Prepucio/citología , Prepucio/virología , Humanos , Masculino , Tonsila Palatina/citología , Tonsila Palatina/virología , Papillomaviridae/fisiología , Transcripción Genética/fisiología , Replicación Viral/fisiologíaRESUMEN
Tobacco, alcohol, and human papillomavirus (HPV) are major risk factors for head and neck cancer (HNC), but it is unclear whether there are two distinct HNC risk groups, one associated with HPV and the other with tobacco/alcohol. Because HPV-positive HNC are clinically distinct from HPV-negative cases in treatment response and with more favorable prognoses, determining whether these differences result from infection alone or in association with other HNC risk factors is important for developing future therapeutic strategies. Incident cases of HNC (n = 201) and age-gender frequency-matched controls (n = 324) were recruited to assess anti-HPV VLP (virus like particles) antibodies 16, 18, 31, and 33. Multivariate logistic regression and stratified analyses were used to calculate adjusted odds ratios (OR). HPV-seronegative and seropositive/heavy tobacco users had similar increased adjusted risks of HNC (HPV-seronegative OR = 2.6, 1.4-5.0; HPV-seropositive OR = 2.3, 1.1-4.8), as did HPV-seronegative (OR = 4.3, 2.1-9.1) versus HPV-seropositive/heavy alcohol users (OR = 3.9, 1.6-9.4). Similar HPV/tobacco/alcohol risk profiles also were seen in oropharyngeal and oral cavity tumor sites. Our finding that tobacco/alcohol use increased the risk of HNC in both HPV-seropositive and HPV-seronegative individuals is consistent with the observation that HPV infection is not a sufficient cause of HNC but requires the accumulation of additional cellular changes.
Asunto(s)
Consumo de Bebidas Alcohólicas/efectos adversos , Neoplasias de Cabeza y Cuello/inducido químicamente , Neoplasias de Cabeza y Cuello/virología , Infecciones por Papillomavirus/complicaciones , Fumar/efectos adversos , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antivirales/sangre , Femenino , Neoplasias de Cabeza y Cuello/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Infecciones por Papillomavirus/epidemiología , Prevalencia , Factores de Riesgo , Adulto JovenRESUMEN
Few large studies have evaluated concordance based on a broad spectrum of human papillomavirus (HPV) types in oral and genital specimens of mothers and their recently born infants. This information is important in determining whether HPV vaccines administered prior to pregnancy may be useful for preventing vertical transmission. HPV DNA was positive in 30% of mothers and 1.5% of newborns. Maternal/newborn concordance (HPV+/+ or HPV-/-) was 71%. Among HPV DNA+ mothers, only 3% of their infants were DNA+ and only 1 pair had the same HPV type. Among HPV- women, 0.8% of infants were HPV+. HPV DNA detected in hospitalized newborns reflects current infection transmitted to infants during pregnancy or delivery. None of the mother/baby HPV DNA+ concordance pairs detected viral types found in HPV vaccines suggesting that vaccination prior to pregnancy is unlikely to be efficacious in preventing vertical transmission.
Asunto(s)
Alphapapillomavirus , Transmisión Vertical de Enfermedad Infecciosa/prevención & control , Infecciones por Papillomavirus/transmisión , Vacunas contra Papillomavirus/administración & dosificación , Infecciones Tumorales por Virus/transmisión , Adulto , Alphapapillomavirus/inmunología , Alphapapillomavirus/aislamiento & purificación , Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , ADN Viral/análisis , Femenino , Papillomavirus Humano 16 , Papillomavirus Humano 18 , Humanos , Recién Nacido , Modelos Logísticos , Infecciones por Papillomavirus/congénito , Infecciones por Papillomavirus/prevención & control , Embarazo , Factores de Riesgo , Infecciones Tumorales por Virus/congénito , Infecciones Tumorales por Virus/prevención & control , Adulto JovenRESUMEN
Interferons (IFNs) have been used to treat mucosal lesions caused by human papillomavirus (HPV) infection, such as intraepithelial precursor lesions to cancer of the uterine cervix, genital warts or recurrent respiratory papillomatosis, to potentially reduce or eliminate replicating HPV plasmid genomes. Mucosal HPVs have evolved mechanisms that impede IFN-beta synthesis and downregulate genes induced by IFN. Here we show that these HPV types directly subvert a cellular transcriptional response to IFN-beta as a potential boost in infection. Treatment with low levels of human IFN-beta induced initial amplification of HPV-16 and HPV-11 plasmid genomes and increased HPV-16 or HPV-31 DNA copy numbers up to 6-fold in HPV-immortalized keratinocytes. IFN treatment also increased early gene transcription from the major early gene promoters in HPV-16, HPV-31 and HPV-11. Furthermore, mutagenesis of the viral genomes and ectopic interferon regulatory factor (IRF) expression in transfection experiments using IRF-1(-/-), IRF-2(-/-) and dual knockout cell lines determined that these responses are due to the activation of IRF-1 interaction with a conserved interferon response element demonstrated in several mucosal HPV early gene promoters. Our results provide a molecular explanation for the varying clinical outcomes of IFN therapy of papillomatoses and define an assay for the modulation of the HPV gene program by IFNs as well as other cytokines and signaling molecules in infection and therapy.
Asunto(s)
Genoma Viral , Papillomavirus Humano 11/genética , Papillomavirus Humano 16/genética , Factor 1 Regulador del Interferón/genética , Interferón beta/farmacología , Replicación Viral , Animales , Inmunoprecipitación de Cromatina , Cartilla de ADN/química , Ensayo de Cambio de Movilidad Electroforética , Femenino , Regulación Viral de la Expresión Génica , Humanos , Factor 1 Regulador del Interferón/metabolismo , Factor 1 Regulador del Interferón/fisiología , Factor 2 Regulador del Interferón/fisiología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/virología , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas/genética , Elementos de Respuesta , Transcripción Genética/efectos de los fármacosRESUMEN
Cellular factors that bind to cis sequences in the human papillomavirus 16 (HPV-16) upstream regulatory region (URR) positively and negatively regulate the viral E6 and E7 oncogene promoter, P97. DNase I footprinting has revealed the binding of cellular proteins to two previously undetected cis elements overlapping and 3' of the transcription-initiation site of the P97 promoter. Mutations within homologous motifs found in both of these cis elements abolished their negative function in vivo and the binding of the same cellular complex in vitro. This factor was identified as YY1 by complex mobility and binding specificity in comparison with vaccinia virus-expressed, purified recombinant YY1 protein and by antigenic reactivity with YY1 antisera. Cis mutations in the 'initiator' YY1 site activated the P97 promoter in vivo and in vitro. P97 was also activated threefold in vitro by depletion of endogenous YY1 with wild-type, but not mutant, YY1 oligonucleotides from the IgH kappa E3' enhancer. Furthermore, increasing concentrations of exogenous, purified recombinant YY1 repressed wild-type P97 transcript levels by up to threefold, but did not influence the P97 promoter mutated in the 'initiator' YY1 site. Thus, the promoter-proximal YY1 site was not necessary for correct transcription initiation at the P97 promoter, but was found to be required for downregulation of P97 transcription in vivo and in vitro. In contrast to other viral and cellular promoters, where YY1 is thought to function as a positive transcription-'initiator' factor, HPV-16 P97 transcription is downregulated by YY1 from a critical motif overlapping the transcription start site.
Asunto(s)
Papillomavirus Humano 16/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Represoras/metabolismo , Sitio de Iniciación de la Transcripción/fisiología , Factor de Transcripción YY1/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo , Regulación Viral de la Expresión Génica/fisiología , Papillomavirus Humano 16/genética , Humanos , Proteínas Oncogénicas Virales/genética , Proteínas Represoras/genéticaRESUMEN
Replication of the double-stranded, circular human papillomavirus (HPV) genomes requires the viral DNA replicase E1. Here, we report an initial characterization of the E1 cistron of HPV type 16 (HPV-16), the most common oncogenic mucosal HPV type found in cervical and some head and neck cancers. The first step in HPV DNA replication is an initial burst of plasmid viral DNA amplification. Complementation assays between HPV-16 genomes carrying mutations in the early genes confirmed that the expression of E1 was necessary for initial HPV-16 plasmid synthesis. The major early HPV-16 promoter, P97, was dispensable for E1 production in the initial amplification because cis mutations inactivating P97 did not affect the trans complementation of E1- mutants. In contrast, E1 expression was abolished by cis mutations in the splice donor site at nucleotide (nt) 226, the splice acceptor site at nt 409, or a TATAA box at nt 7890. The mapping of 5' mRNA ends using rapid amplification of cDNA ends defined a promoter with a transcription start site at HPV-16 nt 14, P14. P14-initiated mRNA levels were low and required intact TATAA (7890). E1 expression required the HPV-16 keratinocyte-dependent enhancer, since cis mutations in its AP-2 and TEF-1 motifs abolished the ability of the mutant genomes to complement E1- genomes, and it was further modulated by origin-proximal and -distal binding sites for the viral E2 gene products. We conclude that P14-initiated E1 expression is critical for and limiting in the initial amplification of the HPV-16 genome.
Asunto(s)
Genes , Papillomavirus Humano 16/genética , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/genética , Secuencia de Bases , Línea Celular , Replicación del ADN , ADN Viral/biosíntesis , Prueba de Complementación Genética , Papillomavirus Humano 16/fisiología , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Sitios de Empalme de ARN , TATA Box , Sitio de Iniciación de la Transcripción , Replicación ViralRESUMEN
A conserved E8(wedge)E2 spliced mRNA is detected in keratinocytes transfected with human papillomavirus type 16 (HPV-16) plasmid DNA. Expression of HPV-16 E8--E2 (16-E8--E2) is independent of the major early promoter, P97, and is modulated by both specific splicing events and conserved cis elements in the upstream regulatory region in a manner that differs from transcriptional regulation of other early viral genes. Mutations that disrupt the predicted 16-E8--E2 message also increase initial HPV-16 plasmid amplification 8- to 15-fold and major early gene (P97) transcription 4- to 5-fold over those of the wild type (wt). Expressing the 16-E8--E2 gene product from the cytomegalovirus (CMV) promoter represses HPV-16 early gene transcription from P97 in a dose-dependent manner, as detected by RNase protection assays. When expressed from the CMV promoter, 16-E8--E2 also inhibits the amplification of an HPV-16 plasmid and a heterologous simian virus 40 (SV40) ori plasmid that contains E2 binding sites in cis. In contrast, cotransfections with HPV-16 wt genomes that express physiologic levels of 16-E8--E2 are sufficient to repress HPV-16 plasmid amplification but are limiting and insufficient for the repression of SV40 amplification. 16-E8--E2-dependent repression of HPV-16 E1 expression is sufficient to account for this observed inhibition of initial HPV-16 plasmid amplification. Unlike with other papillomaviruses, primary human keratinocytes immortalized by the HPV-16 E8 mutant genome contain more than eightfold-higher levels of unintegrated plasmid than the wt, demonstrating that 16-E8(wedge)E2 limits the viral copy number but is not required for plasmid persistence and maintenance.
Asunto(s)
ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Viral de la Expresión Génica , Papillomavirus Humano 16/fisiología , Queratinocitos/virología , Proteínas Oncogénicas Virales/metabolismo , Replicación del ADN , Células HeLa , Humanos , Transcripción Genética , Replicación ViralRESUMEN
Human papillomaviruses (HPV) are associated with nearly all cervical cancers, 20% to 30% of head and neck cancers (HNC), and other cancers. Because HNCs also arise in HPV-negative patients, this type of cancer provides unique opportunities to define similarities and differences of HPV-positive versus HPV-negative cancers arising in the same tissue. Here, we describe genome-wide expression profiling of 84 HNCs, cervical cancers, and site-matched normal epithelial samples in which we used laser capture microdissection to enrich samples for tumor-derived versus normal epithelial cells. This analysis revealed that HPV(+) HNCs and cervical cancers differed in their patterns of gene expression yet shared many changes compared with HPV(-) HNCs. Some of these shared changes were predicted, but many others were not. Notably, HPV(+) HNCs and cervical cancers were found to be up-regulated in their expression of a distinct and larger subset of cell cycle genes than that observed in HPV(-) HNC. Moreover, HPV(+) cancers overexpressed testis-specific genes that are normally expressed only in meiotic cells. Many, although not all, of the hallmark differences between HPV(+) HNC and HPV(-) HNC were a direct consequence of HPV and in particular the viral E6 and E7 oncogenes. This included a novel association of HPV oncogenes with testis-specific gene expression. These findings in primary human tumors provide novel biomarkers for early detection of HPV(+) and HPV(-) cancers, and emphasize the potential value of targeting E6 and E7 function, alone or combined with radiation and/or traditional chemotherapy, in the treatment of HPV(+) cancers.
Asunto(s)
Ciclo Celular/genética , Neoplasias de Cabeza y Cuello/virología , Infecciones por Papillomavirus/genética , Neoplasias del Cuello Uterino/virología , Adulto , Células Epiteliales/patología , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica , Genoma Humano , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Papillomaviridae/genética , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/patología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Regulación hacia Arriba , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patologíaRESUMEN
BACKGROUND: High-risk human papillomavirus (HPV-HR) is a significant risk factor for head and neck cancer (HNC), abrogating normal p53 function. In addition, HPV and p53 have been associated with prognosis of these tumors but the findings have been inconsistent. We examined p53 expression and HPV-HR individually and jointly for differences in predicting HNC survival. METHODS: HNC patients (n = 294) were evaluated for p53 by immunohistochemical staining. HPV was detected by PCR/dot blot hybridization and sequencing. RESULTS: HNC tumors showed 48% with p53 overexpression and 27% with HPV-HR. Multivariate analyses showed that p53 positivity was significantly associated with higher risk of disease-specific [hazard ratio (HR); 2.0; 95% confidence interval (95% CI), 1.1-3.7] and recurrence-free mortality (HR, 2.8; 95% CI, 1.4-5.3). HPV- cases had significantly worse disease-specific survival (HR, 2.8; 95% CI, 1.3-6.3) compared with HPV-HR cases. When analyzed jointly, with p53(-)/HPV-HR tumors as the reference group, p53(+)/HPV(-) patients had the worst disease-specific (HR, 5.3; 58% versus 15%, P = 0.006) and recurrence-free survival rates (HR, 9.5; 17% versus 89%, P = 0.001), in contrast to the p53(-)/HPV(-) and p53(+)/HPV-HR groups, which had less elevated and different risks for disease-specific survival (HR, 2.5 and 1.7, respectively) and recurrence-free survival (HR, 4.2 and 7.2, respectively). CONCLUSION: Joint assessment of p53/HPV status provides different HRs for each clinical outcome in the four biomarker groups that are distinct from the individual biomarkers. These findings suggest that joint assessment of p53/HPV provides a better indicator of prognosis and potentially different types of treatments.
Asunto(s)
Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/virología , Papillomavirus Humano 16/genética , Infecciones por Papillomavirus/complicaciones , Proteína p53 Supresora de Tumor/genética , Biomarcadores de Tumor/genética , Femenino , Neoplasias de Cabeza y Cuello/mortalidad , Papillomavirus Humano 16/aislamiento & purificación , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/genética , Pronóstico , Modelos de Riesgos Proporcionales , Factores de Riesgo , Análisis de SupervivenciaRESUMEN
BACKGROUND: Human papillomavirus (HPV) is a risk factor for head and neck cancers (HNC), yet HPV-associated tumors have better prognosis than HPV-negative tumors. METHODS: We evaluated whether pretreatment presence of antibodies to HPV capsids [virus-like particles (VLP)] or to HPV-16 oncoproteins E6 and E7 was a predictor of HPV-positive HNC and clinical outcomes. Sera from 156 HNC patients were tested for antibodies to HPV-16-derived antigens using ELISA. HPV-16 in tumors was evaluated by PCR and DNA sequencing. RESULTS: HPV-16 antibodies were found in 33% with HPV-16 VLP, 21% with HPV-16 E6, and 21% with E7. HPV-16 was detected in 26% of tumors. There was a strong correlation between detection of HPV-16 tumor DNA and antibodies to HPV-16 E6 or E7 (kappa = 0.7) but not to HPV-16 VLP (kappa = 0.4). Multivariate analyses showed significantly better disease-specific survival in seropositive HPV-16 VLP [hazard ratio (HR), 0.4; 95% confidence interval (95% CI), 0.1-0.9], HPV-16 E6 (HR, 0.1; 95% CI, 0.02-0.5), and HPV-16 E7 (HR, 0.3; 95% CI, 0.1-0.9) cases. Less disease recurrence occurred among those with antibodies to both E6 and E7 compared with those negative to both (P = 0.003). There was better disease-specific survival in patients who were E6 positive at baseline and remained positive at follow-up compared with individuals who were E6 negative at both time points (P = 0.03; kappa = 0.9). CONCLUSIONS: The presence of antibodies to HPV-16 E6 and E7 is associated with HPV in tumor cells and with better clinical outcomes. These findings suggest that the presence of E6/E7 antibodies before treatment is predictive of better clinical outcomes and that they may serve as biomarkers for selecting targeted therapeutic modalities developed for HPV-associated tumors.
Asunto(s)
Anticuerpos Antivirales/sangre , Neoplasias de Cabeza y Cuello/virología , Papillomaviridae/inmunología , Infecciones por Papillomavirus/virología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Neoplasias de Cabeza y Cuello/sangre , Neoplasias de Cabeza y Cuello/terapia , Humanos , Masculino , Persona de Mediana Edad , Proteínas Oncogénicas Virales , Proteínas E7 de Papillomavirus , Infecciones por Papillomavirus/sangre , Reacción en Cadena de la Polimerasa , Pronóstico , Modelos de Riesgos Proporcionales , Proteínas Represoras , Factores de Riesgo , Estadísticas no Paramétricas , Análisis de SupervivenciaRESUMEN
Development of head and neck cancer (HNC) is associated with human papillomavirus high-risk (HPV-HR) types. The HPV E7 oncoprotein inactivates the pRB protein increasing expression of p16(INK4a). P16 expression and HPV status have been associated with differences in clinical outcomes for HNC. This study examined whether HNC prognosis was different when these biomarkers were examined as individual or joint molecular effects. Tumor tissue from 301 HNC patients were analyzed and sequenced for HPV types. P16 was evaluated by immunohistochemical staining. p16 was expressed in 35% and HPV-HR was detected in 27% of HNC patients. After adjustment for age, tobacco, and alcohol, p16+ tumors were statistically significantly associated with HPV-HR (OR=13.3, 7.1-24.9), histology, stage, grade, tumor site, and node involvement. Compared to p16+ HNC cases, those who did not express p16 had significantly worse disease-specific (DS) survival (Hazards Ratio, adj.HR=2.0. 1.0-3.9) and recurrence (adj.HR=3.6, 1.6-8.2); and HPV- cases had worse DS survival (adj.HR=2.8, 1.1-7.1) and recurrence (adj.HR=2.0, 0.8-4.8) compared to HPV-HR patients. Each of the p16/HPV groups had different survival outcomes: p16+/HPV-HR cases (referent) had the best and p16-/HPV- cases had the worst DS survival (adj.HR=3.6; 53% versus 13%, p=0.004) whereas p16+/HPV- and p16-/HPV-HR had similar DS survival (adj.HR=2.7/2.8). p16-/HPV-HR cases had a worse recurrence rate (adj.HR=7.0; 60% versus 0%, referent, p=0.08) than p16-/HPV- (adj.HR=4.5) or p16+/HPV- (adj.HR=1.8) cases. The combined p16/HPV biomarker data are associated with different survival outcomes of HNC compared to each marker evaluated separately, indicating that the two molecular mechanisms evaluated together may provide a more accurate prediction of clinical outcomes.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/virología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/análisis , Neoplasias de Cabeza y Cuello/virología , Papillomavirus Humano 11 , Infecciones por Papillomavirus/complicaciones , Adulto , Anciano , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/cirugía , ADN Viral/análisis , Supervivencia sin Enfermedad , Femenino , Expresión Génica , Neoplasias de Cabeza y Cuello/mortalidad , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/cirugía , Papillomavirus Humano 11/genética , Humanos , Inmunohistoquímica , Antígeno Ki-67/análisis , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/virología , Infecciones por Papillomavirus/mortalidad , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/cirugía , Modelos de Riesgos Proporcionales , Coloración y Etiquetado , Análisis de SupervivenciaRESUMEN
OBJECTIVE: Human papillomavirus (HPV) in the oral cavity or oropharynx is associated with an increased risk of laryngeal papillomatosis, head and neck cancer, and cervical and other genital cancers. We evaluated the prevalence of HPV DNA in the oral cavity/oropharynx in a cross section of children aged 2 weeks to 20 years. METHODS: A risk factor questionnaire and oral exfoliated cells were collected from children (N = 1235). HPV DNA was detected using PCR, dot blot hybridization, and DNA sequencing. RESULTS: The HPV prevalence was 1.9% in the oral cavity/oropharynx of children. A bimodal age distribution was observed with the highest HPV prevalence in the youngest and oldest groups: 2.5% aged <1 year, 0.8% aged 1 to 4 years, 1.2% aged 5 to 11 years, 1.5% in aged 12 to 15 years, and 3.3% in aged 16 to 20 years. The prevalence of the HPV quadrivalent vaccine types (HPV-6, 11, 16, 18) reached 0.9% in the 16- to 20-year age group. In this age group, female gender [odds ratio (OR): 6.9, P = 0.04], genital warts (OR: 19.3, P < 0.01), and current smoker (OR: 6.5, P = 0.01) were associated with a higher risk of being detected with an oral HPV infection. No risk factors in parents were identified with transmission of HPV to infants. CONCLUSIONS: The age-specific prevalence rates of HPV in this large cross section of children and adolescents demonstrate that HPV infection is acquired gradually in childhood. These data support a target age for HPV vaccination before puberty to prevent serious HPV-related genital and oral diseases.
Asunto(s)
Alphapapillomavirus/aislamiento & purificación , Boca/virología , Infecciones por Papillomavirus/epidemiología , Faringe/virología , Adolescente , Adulto , Alphapapillomavirus/clasificación , Niño , Preescolar , Femenino , Hospitales Universitarios , Humanos , Lactante , Recién Nacido , Iowa/epidemiología , Masculino , Prevalencia , Factores de Riesgo , Análisis de Secuencia de ADN , Distribución por Sexo , Encuestas y CuestionariosRESUMEN
We investigated whether herpes simplex viruses, HSV-1 and HSV-2, are cofactors of head and neck cancer (HNC) in association with tobacco, alcohol, or HPV-16 infection. The study included 164 HNC cases and 295 controls. Serologic tests were used to distinguish HSV-1 and HSV-2. Antibodies to anti-VLP HPV-16 and HPV-16 E6 and E7 were evaluated by ELISA. After adjusting for age, tobacco, alcohol use, and number of sexual partners, risk of cancer was not significantly increased in those with HSV-1 [adjusted odds ratio (OR)=0.7] or HSV-2 (OR=0.8) compared to HSV-negative patients. Although heavy use of tobacco, alcohol and HPV-16 infection was associated with an increased risk of HNC, the adjusted risk among those infected with HSV-1 or HSV-2 lowered the odds compared to those who were not infected. Heavy smokers (OR=1.7) and heavy drinkers infected with HSV-1 (OR=4.2) or HSV-2 (smokers: OR=1.6; drinkers: OR=3.2) had lower odds compared to seronegative HSV-1 heavy users (smokers: OR=2.5; drinkers: OR=5.5) or HSV-2 (smokers: OR=1.9; drinkers: OR=6.2). Those seropositive to HPV-16 E6 and/or E7 but not HSV-1 (OR=27.4) or HSV-2 (OR=18.0) had higher risk of HNC compared to those infected with HSV-1 (OR=16.7) or HSV-2 (not estimable). These findings suggest that seropositivity to HSV-1 and HSV-2, although not independent risk factors for HNC, may modify the risk of HNC associated with exposure to tobacco, alcohol, or HPV-HR.
Asunto(s)
Neoplasias de Cabeza y Cuello/virología , Herpes Simple/complicaciones , Herpesvirus Humano 1 , Herpesvirus Humano 2 , Adulto , Anciano , Anciano de 80 o más Años , Consumo de Bebidas Alcohólicas/efectos adversos , Estudios de Casos y Controles , Femenino , Neoplasias de Cabeza y Cuello/etiología , Humanos , Masculino , Persona de Mediana Edad , Factores de Riesgo , Fumar/efectos adversosRESUMEN
In order to examine the association between alcohol dehydrogenase 3 (ADH3) genotypes and risk of head and neck squamous cell carcinomas (HNSCC), we conducted a hospital based case-control study including 348 cases and 330 controls. DNA isolated from exfoliated cells from the oral cavity were genotyped for ADH3 polymorphisms using PCR followed by SspI digestion. Odds ratios (OR) and hazards ratios (HR) were done by unconditional logistic regression and Cox regression. Relative to ADH3(2-2) carriers, ADH3(1-1) [OR, 0.7; 95% confidence interval (CI), 0.4-1.1] and ADH3(1-2) (OR, 0.8; 95% CI, 0.5-1.2) had a nonsignificant reduced risk of HNSCC. ADH(1-2) smokers of >30 pack-years were at decreased risk of oral cavity squamous cell carcinomas compared with ADH3(2-2) (OR, 0.3, 0.1-0.9), whereas ADH3(1-1) smokers were not. After adjustment, those with ADH3(1-2) had significantly worse overall survival compared with ADH3(1-1) (HR, 0.3, 0.2-0.6) or ADH3(2-2) (HR, 0.4, 0.2-0.9) and increased recurrence (ADH3(1-1), 0.2, 0.1-0.6; ADH3(2-2), 0.6, 0.2-1.3). Our data did not show that ADH3 genotypes had a significantly independent effect on the risk of HNSCC, nor did they modify the risks increased by alcohol or tobacco consumption and high-risk human papillomavirus infection. However, participants with ADH3(1-2) genotype were associated with poorer survival compared with those who had the other two ADH3 genotypes and a higher rate of recurrence than participants with ADH3(1-1) genotype.