Loss of HPV16 E2 Protein Expression Without Disruption of the E2 ORF Correlates with Carcinogenic Progression

Integration of the viral DNA in the cellular genome has been suggested to be critical in carcinogenic progression of HPV-associated cervical neoplasia. This event can be accompanied by disruption of the open reading frame (ORF) encoding the E2 repressor, thus leading to transcriptional up-regulation of the E6 and E7 viral oncogenes. At this stage, it is unclear whether disruption of the E2 ORF is mandatory for carcinogenic progression. We measured E2 RNA and protein expression in clinical samples of various grades of HPV16-associated cervical neoplasia and compared it with the status of the viral genome. RNA extracted from paraffin embedded tissues was hybridized to specific probes and quantified by the NanoString technology. Protein expression was appreciated by immunohistochemistry and the status of viral DNA was determined by in situ hybridization, all performed on serial sections of the same samples. E2 protein was found highly expressed in CIN1, CIN2 lesions where the HPV DNA was highly replicative, while it was decreased in more advanced grade lesions where replication is decreased or lost (CIN3 and SCC). In contrast, E2 transcripts could be elevated even in conditions of no or low expression of the protein, as found in the Caski cell line. Our data demonstrate that integration of the viral DNA in the cellular genome does not always lead to disruption of the E2 ORF and drastic reduction of E2 transcripts, while in contrast, expression of the E2 protein is always drastically reduced.