Every year, more than 650,000 new cases of head and neck carcinomas (HNCs) are diagnosed worldwide [1
]. Most HNCs are associated with smoking and alcohol consumption, but nearly 26% of HNCs are linked to infection by high-risk human papillomavirus (HR HPV) [2
]. HPV-driven HNCs are mostly found in the oropharynx, with HR HPV prevalence varying from 7% to 88%, according to the sub-site of oropharyngeal cancer (OPC), the detection method and geographical area [3
]. The incidence of OPC has been increasing markedly over the last decade [4
], and it is widely accepted that HPV-associated HNC represents a different entity. From the clinical point of view, the most important are the improved survival and response to treatment in HPV HNC patients [6
The essential role in the process of viral carcinogenesis is played by two viral oncoproteins, E6 and E7. These viral oncoproteins, via binding to the cellular targets p53 and pRb, cause disruption of cellular growth control pathways [7
]. HPV16–E6/E7 oncogene transcription is initiated from the viral P97 promoter located in the non-coding long control region (LCR), and is fundamentally regulated by the viral E2 protein.
The E2 protein binds to four specific binding sites (E2BS1-4) with a consensus palindromic DNA motif (ACCGN4
CGGT). In the early stages of the viral infection, low-abundance E2 binds to its high-affinity site (E2BS1), which activates viral oncogene transcription and maintains E2 expression. Increasing E2 levels lead to its binding to the repressive low-affinity sites (E2BS3-4) to suppress viral oncogene transcription. The function of E2BS2 is still uncertain (reviewed in Doeberitz and Vinokurova, 2009, McBride and Warburton, 2017 [8
In the permissive viral life cycle, the HPV genome is maintained in an extrachromosomal form. During persistent infection, the HPV genome may integrate into the host genome at fragile and/or transcriptionally-active sites without chromosomal preference. Regarding the viral integrated genome, it is frequently, but not exclusively, interrupted in the E2 ORF, which leads to E2 inactivation. It has been reported that E2 loss results in the disruption of the negative feedback E2 on the E6/E7 oncogene transcription [10
]. The Cancer Genome Atlas (TCGA) analysis of 279 HNC samples revealed also high frequencies of breakpoints in the E1 gene [11
]. As documented from premalignant cervical lesions and cervical cancer (CC), the proportion of cells harboring the integrated genome increases with the disease progression [12
], and patients with the integrated HPV genomes have worse survival rates in comparison to those with the extrachromosomal form of the virus [13
]. In patients with OPC, the presence of an intact E2 ORF was associated with improved clinical outcome in comparison to that in patients with tumors with a disrupted E2 gene [16
], however in larger set of patients, no statistically significant difference was found in disease-specific survival between patients with integrated vs. extrachromosomal and/or mixed forms of HPV [18
]. On the other hand, up to 45% of CC and 51% of HNC tumors harbor only extrachromosomal HPV genomes [12
], which demonstrates that integration is a frequent but not necessary step in HPV-initiated cancer progression.
Disruption of the E2 gene has been correlated with a higher expression of viral E6/E7 mRNA in the cervical cancer cell line [21
], but the results obtained from patients’ samples of CC as well as OPC are inconsistent with in vitro observation [19
]. The viral load has been shown to correlate with disease severity and E6 and E7 oncogene expression in cervical cancer [25
]. However, in HNC and HNC-derived cell lines, no association was found between viral load, integration and viral oncogene expression [23
Epigenetic modifications have been proposed as another possible mechanism for E2 inactivation [28
]. CpG methylation at the repressive E2BSs may disable E2 binding and prevent its repressive function on E6/E7 oncogene transcription. The methylation levels of the E2BS1, -3 and -4 were found to be higher in CC with extrachromosomal compared to integrated HPV genomes [28
]. This phenomenon has also been observed for the repressive E2BSs in HPV-driven oropharyngeal cancers [31
]. Another possible mechanism of disrupting the negative feedback of E2 in E6/E7 transcription could be mutations in the repressive E2BS3 or E2BS4 that make this regulation ineffective.
In this study, we searched for the mechanism by which HPV16 present in the tonsillar carcinoma tissues in an extrachromosomal form contributes to the malignant transformation of the cell. We compared the methylation levels, mutation frequencies in E2BSs, presence of nucleotide variants in LCR and the complete E2 gene, viral load and expression levels of viral oncogenes E6 and E7 in HPV-driven tonsillar carcinomas with the extrachromosomal or integrated virus genomes.
Integration of the HPV genome into the cell genome is an important but not essential step in virus-driven carcinogenesis in cervical and head and neck carcinomas. The integration imparts a selective growth advantage through an enhanced expression and higher stability of fusion virus–host transcripts [10
]. Also, it seems that particular cell clones are selected during the transformation process according to the site of integration. These clones are characterized by increased proliferation and improved survival [11
]. HPV integration itself is directly associated with genetic instability and extensive host genomic amplifications and rearrangements [40
]. However, in many HPV-driven tumors, the integration of viral genomes into host chromosomes were not detected, and the cancer initiation mechanism is still not fully elucidated. To clarify the mechanisms of carcinogenesis driven by the extrachromosomal form of HPV as well as by the mixed form with expression of E2, we performed complex analyses of 18 tonsillar tumor samples with HPV16 active infection. LCR and E2 ORFs single nucleotide variant analysis, determination of CpG methylation level of E2BSs, viral load evaluation and viral oncogene E6/E7 transcript quantification, were done. The E2 mRNA detection using mapping of the viral transcripts was chosen as the selective marker for our analysis, not only as markers of the extrachromosomal form of HPV, but also for the role of E2 in the viral life cycle. The E2 protein negatively regulates the expression of viral oncoproteins E6/E7, the main drivers of viral carcinogenesis, and is also important together with E1 protein for viral replication.
Methylation of CpGs in E2BSs has been shown, in vitro, to affect the binding affinity of the E2 protein, to inhibit its repressive function, and to enhance the expression of the E6 and E7 oncoproteins [29
]. Higher methylation levels in E2BS3-4 have been found in CC with extrachromosomal HPV DNA [19
]. Among the OPC samples, three groups with different methylation levels of E2BSs have been identified; complete methylation of E2BS3 and -4 have been associated with the presence of integrated HPV genomes with an intact E2 gene. In these samples, multiple viral integrated copies were present (tandem integration). An intermediate methylation level (20%–80%) was identified in samples with an extrachromosomal form of the HPV genome, and no methylation was detected in samples with the integrated HPV genome with a disrupted E2 gene [31
]. Compared to previous studies, our results showed significantly higher methylation levels of the repressive E2BS2-4 in the samples with the integrated HPV genome where no E2 mRNA was detected, but the level of methylation of LCR in our study did not affect the level of expression of viral E6/E7 mRNA. A similar observation was made by Park et al. (2011) [41
]. This can be explained by the results of Hatano et al. (2017), who found that the methylation of the HPV genome and human region near the integration site display the same methylation pattern [42
]. So, in comparison to the extrachromosomal form of the viral genome, the methylation level of the integrated form is likely to be more influenced by the site of integration. Moreover, in the present study, the analyzed level of methylation was restricted to E2BSs, and therefore we cannot exclude the possibility that E6/7 oncogene expression is influenced by altered methylation at other HPV genome regions.
The E2 binding affinity to repression sites in LCR may be also affected by mutations. We found no mutation within E2BSs in any sample harboring either the integrated or extrachromosomal form of the HPV genome. The sequence variation in the YY1 binding site was observed in both groups tested without any difference between groups. However, sequence analysis revealed one frequent polymorphism (7518 G to A) in the binding site for the YY1 transcription factor acting as a repressor of HPV P97 transcription [43
]. This polymorphism was previously found in CC samples more frequently in comparison to HPV16 isolates from healthy women [44
]. We evaluated the E6*I mRNA level in tonsillar tumor samples with or without G to A transition, and detected no significantly higher transcription levels in samples with the variant type of 7518 nucleotide. Therefore, the influence of sequences variation in the YY1 sites on transcription of the viral genome needs to be further explored.
The whole E2 gene was identified in the majority of our samples. No mutation leading to protein truncation was found. Most of samples belongs to the A variant lineage and only one sample to the D variant, which was determined by comparison with the HPV16 reference sequence both for the A and D lineages. No specific variants that may represent the other mechanisms of E2 inactivation were identified in samples with the extrachromosomal form of the HPV genome.
We observed no statistically significant difference in the viral load between the extrachromosomal/mixed and integrated genome status. The viral load was slightly, although not significantly, higher in samples with the extrachromosomal/mixed genome form. Similar data were reported by researchers examining CC [19
] or HNC [16
], but other studies did not find any differences in the viral loads [19
]. When comparing the levels of expression of E6 transcripts, the E6*I transcript was more abundant than the E6 FL transcript. No increase of E6/E7 mRNA was observed in those samples with the integrated HPV genome without E2 mRNA, which is in agreement with the results of other studies on CC and HNC cancer [23
]. Increasing levels of the E6/E7 transcripts were detected in premalignant cervical lesions, with the highest level being reported in invasive cancers [48
], and this level seems not to be influenced by other factors, and to be sufficient for the maintenance of the malignant phenotype.