Human Papillomavirus Type 16 (HPV-16) Genomes Integrated in Head and Neck Cancers and in HPV-16-Immortalized Human Keratinocyte Clones Express Chimeric Virus-Cell mRNAs Similar to Those Found in Cervical Cancers

Department of Pathology, University of Iowa Roy J and Lucille A Carver College ofMedicine, Veterans Affairs Medical Center, Iowa City, Iowa 52242, USA.
Journal of Virology (Impact Factor: 4.44). 02/2011; 85(4):1645-54. DOI: 10.1128/JVI.02093-10
Source: PubMed


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.

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Available from: Jens Peter Klussmann, Feb 02, 2014
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    • "Using Amplification of Papillomavirus Oncogene Transcripts PCR (APOT-PCR), so far only two studies report HPV16 integration in 2 out of 4 and 6 out of 9 tumors in HPV-DNA positive OSCC [13],[14]. "
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    ABSTRACT: Infection with high-risk human papillomavirus (HPV) type 16 is an independent risk factor for the development of oropharyngeal squamous cell carcinomas (OSCC). However, it is unclear whether viral integration is an essential hallmark in the carcinogenic process of OSCC and whether HPV integration correlates with the level of viral gene transcription and influences the expression of disrupted host genes. We analyzed 75 patients with OSCC. HPV16-positivity was proven by p16(INK4A) immunohistochemistry, PCR and FISH. Viral integration was examined using DIPS- as well as APOT-PCR. Viral E2, E6 and E7 gene expression levels were quantified by quantitative reverse transcriptase (RT-q)PCR. Expression levels of 7 human genes disrupted by the virus were extracted from mRNA expression profiling data of 32 OSCCs. Viral copy numbers were assessed by qPCR in 73 tumors. We identified 37 HPV16-human fusion products indicating viral integration in 29 (39%) OSCC. In the remaining tumors (61%) only episome-derived PCR products were detected. When comparing OSCC with or without an integration-derived fusion product, we did not find significant differences in the mean RNA expression of viral genes E2, E6 and E7 or the viral copy numbers per cell, nor did the RNA expression of the HPV-disrupted genes differ from either group of OSCC. In conclusion, our data do not support the hypothesis that integration affects the levels of viral and/or HPV-disrupted human gene transcripts. Thus constitutive, rather than a high level, of expression of oncogene transcripts appears to be required in HPV-related OSCC.
    PLoS ONE 02/2014; 9(2):e88718. DOI:10.1371/journal.pone.0088718 · 3.23 Impact Factor
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    • "; Lace et al. 2011; Khoury et al. 2013). Prior data, based on SNP arrays, suggested that approximately one-third of HPV integrants in cervical cancers were statistically associated with regional CNVs (Peter et al. 2010). "
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    ABSTRACT: Genomic instability is a hallmark of human cancers, including the 5% caused by human papillomavirus (HPV). Here we report a striking association between HPV integration and adjacent host genomic structural variation in human cancer cell lines and primary tumors. Whole genome sequencing revealed HPV integrants flanking and bridging extensive host genomic amplifications and rearrangements, including deletions, inversions and chromosomal translocations. We present a model of 'looping' by which HPV integrant-mediated DNA replication and recombination may result in viral-host DNA concatemers, frequently disrupting genes involved in oncogenesis and amplifying HPV oncogenes E6 and E7. Our high-resolution results shed new light on a catastrophic process, distinct from chromothripsis and other mutational processes, by which HPV directly promotes genomic instability.
    Genome Research 11/2013; 24(2). DOI:10.1101/gr.164806.113 · 14.63 Impact Factor
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    • "Several studies have provided evidence, that integration of high risk HPV types, such as HPV16 and HPV18, is a pivotal step in the transition of cervical intraepithelial neoplasia (CIN) to invasive carcinoma [27], [35]. Also in OSCC, HPV16 is often found integrated [17], [26], [36], [37]. In contrast, the low risk HPV types 6 and 11 are mostly present in the episomal form, both in uterine benign and CIN-lesions as well as in laryngeal papilloma [34], [38]. "
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    ABSTRACT: Juvenile-onset recurrent respiratory papillomatosis (RRP) is associated with low risk human papillomavirus (HPV) types 6 and 11. Malignant transformation has been reported solely for HPV11-associated RRP in 2-4% of all RRP-cases, but not for HPV6. The molecular mechanisms in the carcinogenesis of low risk HPV-associated cancers are to date unknown. We report of a female patient, who presented with a laryngeal carcinoma at the age of 24 years. She had a history of juvenile-onset RRP with an onset at the age of three and subsequently several hundred surgical interventions due to multiple recurrences of RRP. Polymerase chain reaction (PCR) or bead-based hybridization followed by direct sequencing identified HPV6 in tissue sections of previous papilloma and the carcinoma. P16(INK4A), p53 and pRb immunostainings were negative in all lesions. HPV6 specific fluorescence in situ hybridization (FISH) revealed nuclear staining suggesting episomal virus in the papilloma and a single integration site in the carcinoma. Integration-specific amplification of papillomavirus oncogene transcripts PCR (APOT-PCR) showed integration in the aldo-keto reductase 1C3 gene (AKR1C3) on chromosome 10p15.1. ArrayCGH detected loss of the other gene copy as part of a deletion at 10p14-p15.2. Western blot analysis and immunohistochemistry of the protein AKR1C3 showed a marked reduction of its expression in the carcinoma. In conclusion, we identified a novel molecular mechanism underlying a first case of HPV6-associated laryngeal carcinoma in juvenile-onset RRP, i.e. that HPV6 integration in the AKR1C3 gene resulted in loss of its expression. Alterations of AKR1C gene expression have previously been implicated in the tumorigenesis of other (HPV-related) malignancies.
    PLoS ONE 02/2013; 8(2):e57207. DOI:10.1371/journal.pone.0057207 · 3.23 Impact Factor
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