Differences in Stability of Viral and Viral-Cellular Fusion Transcripts in HPV-Induced Cervical Cancers.

HPV-DNA integration results in dysregulation of viral oncogene expression. Because viral-cellular fusion transcripts inherently lack the viral AU-rich elements of the 3'UTR, they are considered to be more stable than episome-derived transcripts. The aim of this study is to provide formal proof for this assumption by comparing the stability of viral early transcripts derived from episomal and integrated HPV16 DNA, respectively. Full-length cDNA of three fusion transcripts comprising viral and cellular sequences in sense orientation were amplified and cloned into the adeno-viral-vector pAd/CMV/V5-DEST. The most abundant HPV16 oncogene transcript E6*I-E7-E1vE4-E5 with and without 3'UTR, served as reference and control, respectively. Human primary keratinocytes were transduced using high titer virus stocks. qRT-PCR was performed to determine mRNA stability in relation to GAPDH in the presence of actinomycin-D. In four independent transduction experiments, all three viral-cellular fusion transcripts were significantly more stable compared to the episome-derived reference. Among the three viral-cellular fusion transcripts the most stable transcript was devoid of the instability core motif "AUUUA". Unexpectedly, there was no significant difference in the stability between the episome-derived transcripts either with or without 3'UTR, indicating that the AU-rich elements of the 3'UTR are not contributing to RNA stability. Instead, the three "AUUUA" motifs located in the untranslated region between the viral E4 and E5 genes may be responsible for the instability. This is the first report showing that authentic viral-cellular fusion transcripts are more stable than episome-derived transcripts. The longer half-life of the fusion transcripts may result in increased levels of viral oncoproteins and thereby drive the carcinogenic process.

Non-random integration of the HPV genome in cervical cancer.

HPV DNA integration into the host genome is a characteristic but not an exclusive step during cervical carcinogenesis. It is still a matter of debate whether viral integration contributes to the transformation process beyond ensuring the constitutive expression of the viral oncogenes. There is mounting evidence for a non-random distribution of integration loci and the direct involvement of cellular cancer-related genes. In this study we addressed this topic by extending the existing data set by an additional 47 HPV16 and HPV18 positive cervical carcinoma. We provide supportive evidence for previously defined integration hotspots and have revealed another cluster of integration sites within the cytogenetic band 3q28. Moreover, in the vicinity of these hotspots numerous microRNAs (miRNAs) are located and may be influenced by the integrated HPV DNA. By compiling our data and published reports 9 genes could be identified which were affected by HPV integration at least twice in independent tumors. In some tumors the viral-cellular fusion transcripts were even identical with respect to the viral donor and cellular acceptor sites used. However, the exact integration sites are likely to differ since none of the integration sites analysed thus far have shown more than a few nucleotides of homology between viral and host sequences. Therefore, DNA recombination involving large stretches of homology at the integration site can be ruled out. It is however intriguing that by sequence alignment several regions of the HPV16 genome were found to have highly homologous stretches of up to 50 nucleotides to the aforementioned genes and the integration hotspots. One common region of homologies with cellular sequences is between the viral gene E5 and L2 (nucleotides positions 4100 to 4240). We speculate that this and other regions of homology are involved in the integration process. Our observations suggest that targeted disruption, possibly also of critical cellular genes, by HPV integration remains an issue to be fully resolved.

Loss of gene function as a consequence of human papillomavirus DNA integration.

Integration of the human papillomavirus (HPV) genome into the host chromatin is a characteristic step in cervical carcinogenesis. Integration ensures constitutive expression of the viral oncogenes E6 and E7 which drive carcinogenesis. However, integration has also an impact on host DNA. There is increasing evidence that integration not only occurs in fragile sites and translocation breakpoints but also in transcriptionally active regions. Indeed, a substantial number of integration sites actually disrupt host genes and may thereby affect gene expression. No doubt, even subtle changes in gene expression may influence the cell phenotype but small fold changes are difficult to quantify reliably in biopsy material. We have, therefore, addressed the question whether a complete loss of gene function that is insertional mutagenesis in combination with deletion or epigenetic modification of the second allele is also a phenomenon pertinent to cervical cancer. Out of the ten preselected squamous cell carcinomas analyzed, all viral integration sites were located within the intron sequences of known genes, giving rise to viral-cellular fusion transcripts of sense orientation. Moreover, for two tumors, we provide evidence for complete functional loss of the gene affected by HPV integration. Of particular note is that one of the genes involved is the recently described novel tumor suppressor gene castor zinc finger 1. Although our study provides no functional proof that any of the genes affected by HPV integration are causally involved in the transformation process, an exhaustive systematic look at the role of insertional mutagenesis in cervical cancer appears to be warranted.

The majority of viral-cellular fusion transcripts in cervical carcinomas cotranscribe cellular sequences of known or predicted genes.

Integration of human papillomavirus (HPV) DNA into the host genome is a frequent event in cervical carcinogenesis and is reported to occur at randomly selected chromosomal sites. However, as the databases are being up-dated continuously, the knowledge based on sequenced viral integration sites also expands. In this study, viral-cellular fusion transcripts of a preselected group of 74 cervical carcinoma or cervical intraepithelial neoplasia grade 3 (CIN3) biopsies harboring integrated HPV16, HPV18, HPV31, HPV33, or HPV45 DNA were amplified by 3'-rapid amplification of cDNA ends PCR and sequenced. Consistent with previous reports, integration sites were found to be distributed throughout the genome. However, 23% (17 of 74) of the integration sites were located within the cytogenetic bands 4q13.3, 8q24.21, 13q22.1, and 17q21, in clusters ranging from 86 to 900 kb. Of note is that clusters 8q24.21 and 13q22.1 are within 1.5 Mbp of an adjacent fragile site whereas clusters 4q13.3 and 17q21 are >15 Mbp distant to any known fragile sites. It is tempting to speculate that as yet unknown fragile sites may be identified on the basis of HPV integration hotspots. No correlation between HPV type and specific integration loci was found. Of 74 fusion transcripts, 28 contained cellular sequences, which were homologous to known genes, and 40 samples contained sequences of predicted genes. In 33 fusion transcripts, both viral and cellular sequences were in sense orientation, indicating that the gene itself or upstream sequences were affected by integration. These data suggest that the influence of HPV integration on host gene expression may not be a rare effect and should encourage more detailed analyses.

Type-dependent integration frequency of human papillomavirus genomes in cervical lesions.

Chromosomal integration of high-risk human papillomavirus (HR-HPV) genomes is believed to represent a significant event in the pathogenesis of cervical cancer associated with progression from preneoplastic lesions to invasive carcinomas. This hypothesis is based on experimental data suggesting that integration-dependent disruption of HR-HPV E2 gene functions is important to achieve neoplastic transformation and on clinical data gathered by analyzing lesions induced by human papillomavirus (HPV) 16 and 18 that revealed integrated viral genome copies in the vast majority of cervical cancer cells. However, a substantial fraction of cervical cancers is associated with other HR-HPV types for which virtually no data concerning their integration status have been reported so far. Here, we compared integration frequencies of the five most common oncogenic HPV types (HPV16, 18, 31, 33, and 45) in a series of 835 cervical samples using a specific mRNA-based PCR assay (Amplification of Papillomavirus Oncogene Transcripts). Most precancerous lesions displayed exclusively episomal viral genomes, whereas 62% of the carcinomas had integrated viral genomes. However, the frequency of integrated HR-HPV genomes showed marked differences for individual HR-HPV types. HPV16, 18, and 45 were found substantially more often in the integrated state compared with HPV types 31 and 33. The analysis of the median age of patients with high-grade precancerous lesions and invasive cancers suggests that precancers induced by HPV types 18, 16, and 45 progress to invasive cervical cancer in substantially less time compared with precancers induced by HPV types 31 and 33. These findings suggest that integration of oncogenic HPV genomes in cervical lesions is a consequence rather than the cause of chromosomal instability induced by deregulated HR-HPV E6-E7 oncogene expression. Distinct HR-HPV types apparently provoke chromosomal instability in their host cells to a different extent than is reflected by their integration frequencies in advanced lesions and the time required for CIN 3 lesions to progress to invasive cancer.