Context: Cervical cancer is a rare complication of persistent infection with high risk Human Papillomavirus (HPV) of which HPV16 is the most carcinogenic. HPV genome consists in a ds circular DNA with two regions encoding: i) early “E” and late “L” proteins and ii) a Long Control Region (LCR) involved in viral cycle and early gene regulation. HPV DNA integration in the host cell genome, a frequent but not mandatory event in cervical carcinogenesis, leads to the loss of the E2 gene and consequently to E2 protein. E2 normally binds to E2 Binding Sites (E2BS) present on the LCR and represses transcription of E6 and E7 oncoproteins. Therefore, a loss of E2 induces an overexpression of E6 and E7 proteins that thus induce p53 and pRb ubiquitination and their degradation. Since CpG dinucleotides are present in HPV16 E2BS, we investigated whether E6 HPV16 expression is also submitted to epigenetic regulation.
Materiel and Methods: CaSki cells (integrated HPV16 DNA, methylated E2BS) and SiHa cells (integrated HPV16 DNA, unmethylated E2BS) were treated by 2.5 μM of 5-aza-2′-deoxycytidin (5azadC) for 48h. Furthermore, CasKi cells were transfected with pciNeoE2 to restore E2 expression. A chromatin immunoprecipitation (ChIP) assay with anti-E2 and control antibodies was then performed in CaSki cells transfected or not with pciNeoE2 to control whether E2BS demethylation promotes the binding of E2 on E2BS.
Results: A decrease of E2BS methylation was observed following treatment of CaSki cells with 5azadC. This was accompanied by a decrease of E6 expression both at the mRNA and protein levels as determined by RTqPCR and western-blotting. The decrease of E6 expression was even more significant when E2 was transfected in CaSki cells treated by 5azadC. We found using a ChIP experiment with an anti-E2 Ab, that a 5azadC treatment of E2 expressing CaSki cells induced a 2-fold enrichment of E2BS containing fragments. This clearly indicates that viral DNA demethylation promoted the binding of E2 on the viral promoter.
Unexpectedly, a decrease expression of E6 was also documented in SiHa cells treated by 5azadC. Thus, we hypothesized that cellular factors, themselves submitted to epigenetic regulation, could modulate viral oncoprotein expression. Among candidates, Sp1 able to bind Sp1BS also present on HPV16 promoter is unlikely to be involved as its expression is increased in both CaSki and SiHa cells treated by 5azadC. In contrast, TBX20, a transcriptional factor related to TBX2 himself known to repress HPV16 promoter, is a promising candidate as its expression is highly increased by 5 azadC in both cell lines.
Conclusion: Taken as a whole our data demonstrate that HPV16 oncoprotein expression is regulated in an epigenetic manner via viral and cellular factors. HPV-associated carcinogenesis is not yet fully understood and deciphering new molecular mechanisms involved in tumor development gives opportunities to identify new biomarkers and therapeutic targets.
Citation Format: Adrien Morel, Aurélie Baguet, Caroline Demeret, Eric Hervouet, Christiane Mougin, Jean-Luc Prétet. Epigenetic regulation of HPV16 early genes in a model of cervical cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2717. doi:10.1158/1538-7445.AM2015-2717