Molecular Mechanisms of Loss of E7 Expression in HPV16 – Transformed Human Keratinocytes

Human papillomavirus (HPV) causes about 5% of all human cancers. The HPV oncoproteins E6/E7 are responsible for the transforming potential of the virus. Although continuous expression of the HPV oncogenes was considered indispensable for HPV-induced carcinogenesis, we and others have demonstrated that in a subset of HPV-positive head and neck and cervical cancers, the HPV oncogenes are not expressed (HPV-inactive cancers). Based on the observation that primary HPV-positive tumors express E6/E7, while metastases tend to be HPV-inactive, we hypothesized that HPV-inactive cancers begin as HPV-active lesions and lose their dependence on continuous E6/E7 expression during progression. This may be due to genetic and/or epigenetic modifications caused by the genomic instability and the additional carcinogens to which the tumor is exposed. We observed that HPV-inactive cancers of the cervix often have mutated p53, while HPV-active cancers do not. Therefore, we proposed that HPV positive tumors may become inactive if p53 becomes mutated. The CRISPR-Cas9 system was used to knock-out the p53 gene in differentiation-resistant HPV16 immortalized human keratinocytes (HKc-DR). The DNA deletions within the p53 gene were confirmed by PCR and gel electrophoresis and further validated by Sanger sequencing. Using qPCR, we found that HPV16 E7 expression was significantly lower (5-fold) in the p53-knocked out (KO) lines than in the p53-wild vi type (WT) lines. Reduced E7 expression in p53-KO lines was reversed by using the demethylating agent 5-Aza-2’-deoxycytidine, suggesting that DNA methylation plays a role in this process. Also, we used in situ hybridization to detect HPV16 E7 mRNA in p53-WT and KO lines grown as spheroids on an agarose cushion. Interestingly, while all p53-WT lines have a uniform distribution of E7 signal, the p53-KO lines showed some spheroids that were completely lacking E7 mRNA, and some had a mixed population of E7-positive and E7-negative cells. These results indicate that the p53-KO lines are a heterogeneous population in regard to HPV16 E7 expression. We concluded that p53 loss-of-function mutation may be an important factor in driving HPV16 transformed cells to lose dependence on the continuous expression of the HPV oncogenes and become HPV-inactive. However, complete loss of p53 alone is not sufficient to suppress E7 expression entirely. We also determined that loss of E7 expression may be due, at least in part, to DNA methylation. We are currently examining HPV URR methylation in the p53-WT and KO lines, and isolating pure lines with complete loss of HPV16 E7 expression for further studies of the molecular mechanisms that may lead to the HPV-inactive phenotype.