Positive markers of epithelial-mesenchymal transition (EMT) in head and neck cancers complicate clinical management and are associated with reduced survival. We discuss recent translational discoveries in EMT and suggest additional actionable molecular pathways, biomarkers, and clinical agents. Electronic supplementary material The online version of this article (doi:10.1186/s40169-014-0039-9) contains supplementary material, which is available to authorized users.
High-risk human papillomaviruses (HPV) cause 5% of all human cancers and are primary etiologic agents of cervical, anal, and oropharyngeal cancer. HPV infection is necessary, but not sufficient per se to produce cancer: additional changes must occur that transform HPV-infected cells to malignancy. The HPV oncoproteins E6 and E7 immortalize human keratinocytes, cervical cells, and fibroblasts in culture. Each oncoprotein interacts with a variety of cellular binding partners; most important for transformation are E6 and E7's interactions with p53 and RB (respectively) which lead to degradation of p53 and RB through the ubiquitin pathway. Inactivation of p53 and RB leads to inactivation of pivotal cell cycle checkpoints, thereby stimulating cell proliferation and allowing cell division to occur independently of the presence of DNA damage, replicative stress, and other such insults, leading to genome instability. Continuous expression of E6/E7 drives the proliferation and progression of most HPV-mediated cancers of the cervix and a substantial fraction of those of the oropharynx. However, at both cancer sites, "HPV-inactive" tumors that contain HPV DNA, but do not express E6/E7 arise. We propose that these HPV-inactive cancers begin as HPV-driven lesions, but lose E6/E7 expression at some point during progression. We have recently shown that p53 deletion in HPV-immortalized, premalignant cells allows for the emergence of cell populations that no longer express E6/E7. These findings corroborate the notion of a pivotal role of p53 in the context of HPV-mediated transformation, both at the initiation and progression stages of cancer development.
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 upon 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 knock out (KO) lines than in the p53 wild 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 heterogenous population with 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 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. Citation Format: Fadi F. Abboodi, Kim E. Creek, Lucia A. Pirisi. Molecular mechanisms of loss of E7 expression in HPV16-transformed human keratinocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-329.
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