Differential methylation of E2 binding sites in episomal and integrated HPV 16 genomes in preinvasive and invasive cervical lesions

Abstract

Enhanced expression of the HPV 16 E6-E7 oncogenes may trigger neoplastic transformation of the squamous epithelial cells at the uterine cervix. The HPV E2 protein is a key transcriptional regulator of the E6-E7 genes. It binds to four E2 binding sites (E2BSs 1-4) in the viral upstream regulatory region (URR). Modification of E2 functions, for example, by methylation of E2BSs is hypothesized to trigger enhanced expression of the viral E6-E7 oncogenes. In the majority of HPV-transformed premalignant lesions and about half of cervical carcinomas HPV genomes persist in an extra-chromosomal, episomal state, whereas they are integrated into host cells chromosomes in the remaining lesions. Here we compared the methylation profile of E2BSs 1-4 of the HPV 16 URR in a series of 18 HPV16-positive premalignant lesions and 33 invasive cervical cancers. CpGs within the E2BSs 1, 3, and 4 were higher methylated in all lesions with only episomal HPV16 genomes compared with lesions displaying single integrated copies. Samples with multiple HPV16 integrated copies displayed high methylation levels for all CpGs suggesting that the majority of multiple copies were silenced by extensive methylation. These data support the hypothesis that differential methylation of the E2BSs 1, 3 and 4 is related to the activation of viral oncogene expression in cervical lesions as long as the viral genome remains in the episomal state. Once the virus becomes integrated into host cell chromosomes these methylation patterns may be substantially altered due to complex epigenetic changes of integrated HPV genomes.Persistent infections with high-risk human papillomaviruses (HR-HPVs) are the primary cause of cancer at various sites of the anogenital tract, including the cervix, vulva, vagina, penis and anus, as well as a subset of head and neck cancers. 1 The predominant HR-HPV type in cervical cancers is HPV16, accounting alone for about 50% of all cervical cancers. 2 It is commonly accepted that HPVs infect the basal cells of the squamous epithelium. Oncogenic human papillomaviruses encode two well-characterized oncoproteins, E6 and E7 that are under tight transcriptional control in early permissive, but not yet transformed genital HPV-infections. The enhanced expression of these oncogenes in basal and parabasal squamous epithelial cells of the infected epithelium is the cause of neoplastic transformation of the infected cells. 3,4 The main transforming properties of the E6 and E7 oncoproteins are related to their ability to inactivate the p53 and retinoblastoma (pRB) tumor suppressor proteins, respectively. 4-7 E7 overexpression in HPV-infected cells is accompanied by substantial overexpression of the cyclin-dependent kinase inhibitor p16 INK4a . p16 INK4a overexpression is therefore used as surrogate biomarker for HPV-transformed epithelial cells. 8 Expression of the E6 and E7 genes is regulated by the upstream regulatory region (URR). The HPV 16 URR contains sequences that regulate transcription and replication of the viral genome to which both cell...

Methylation status of HPV16 E2‐binding sites classifies subtypes of HPV‐associated oropharyngeal cancers

“…1), was quantitatively analyzed by bisulfite pyrosequencing as described earlier. 17 In brief, tumor tissue was manually microdissected from formalin-fixed, paraffin-embedded tissue sections. Cell lines were used directly after harvest.…”

“…Three primer pairs covering the distal E2BS1 and E2BS2 and the proximal E2BS3 and E2BS4 were used for amplification. 17 Pyrosequencing was performed using the PyroMarkTM Q24 instrument (Qiagen) according to the manufacturer's protocol. Assay setup and sequence run, as well as analysis, were performed using the PyroMarkTM Q24 Software.…”

“…15 However, several findings have strongly suggested that as well as integration-mediated E2 gene disruption, other mechanisms may contribute to the inhibition of E2 functions. First, it was reported that in approximately 50% of HPV16-associated cervical cancers 16,17 and OPSCC 18,19 only episomal HPV genomes and no integration-mediated disruption of the E2 gene was observed. Second, a substantial percentage of cell lines and tumors with integrated HPV genomes carry additional viral genomes with intact E2 gene sequences, either as episomes or integrated head-to-tail concatemers.…”

Polymorphisms and Functional Analysis of the Intact Human Papillomavirus16 E2 Gene

“…This observation suggested that other mechanisms besides an integration of the viral genome into host cell chromosomes may contribute to the deregulation of the E6-E7 oncogenes during the transition into the transforming mode of HPV infections. Such mechanisms may include de novo methylation or mutations of E2BSs that might cause up-regulation of 10256 E6-E7oncogene expression by impairing the ability of E2 to bind to its specific sites (Romanczuk et al, 1990;Nishimura et al, 2000;Bhattacharjee and Sengupta, 2006;Chaiwongkot et al, 2013;Jacquin et al, 2013).…”

“…Chaiwongkot et al reported that CpGs within the E2BSs 1, 3, and 4 were more highly methylated in all cervical lesions with only episomal HPV16 genomes rather than lesions displaying single integrated copies and suggested that differential methylation of these E2BSs is related to the activation of viral oncogene expression in cervical lesions as long as the viral genome remains in the episomal state (Chaiwongkot et al, 2013). Genetic variations at E2BSs have been shown by previous mutational and functional experiments that could inhibit the binding of E2 protein on its binding site.…”