Hypermethylation of both the viral and host genome is a frequent event during HPV-induced carcinogenesis. Specifically, methylation in the binding sites of the HPV E2 protein (E2BS) inducing HPV E6/E7 oncogene overexpression and of tumor suppressor gene promoters have prompted the concept that demethylating agents could represent a novel therapeutic approach against HPV-induced (pre-)cancerous lesions. Previous experiments in our department demonstrated a dose-dependent reversal of the malignant phenotype upon short-term treatment of HPV-transformed cells with the demethylating agent 5-aza-2’-deoxycytidine (DAC). We sought to analyze DAC-induced effects on HPV-transformed cervical cells in greater detail applying long-term DAC treatment in 2-dimensional cell culture. Further, we analyzed consequences of long-term DAC treatment on cell proliferation and tumor cell invasiveness in 3-dimensional tumor models of HPV-transformed cells.
HPV-transformed cell lines (CaSki, SiHa) were treated over a period of one and two weeks with different concentrations (0.1, 0.5, and 1.0 µM) of the demethylating agent DAC. Expression of the viral oncogenes was determined by qPCR (E6*I, E7) and Western Blot (E7). p16INK4a and p53 protein levels were analyzed by Western Blot. β-galactosidase staining was performed to determine the induction of cellular senescence in treated cells. Tumor spheroids of CaSki and SiHa cells were established, treated with different concentrations of DAC over a period of one and two weeks, respectively, and cellular proliferative activity was analyzed using EdU immunofluorescence. A three-dimensional organotypic culture (OTC) model of CaSki cells was established, treated with DAC and analyzed for proliferative activity and tumor cell invasiveness applying p16INK4a/Ki-67 immunohistochemistry (IHC).
We observed a dose-dependent significant reduction of cellularity and reversal of the malignant cell phenotype characterized by significant down-regulation of viral oncogene and p16INK4a expression, up-regulation of p53 protein levels and induction of cellular senescence measured by β-galactosidase staining in treated CaSki and SiHa cells. All effects were even further pronounced in the second week of treatment. We could further demonstrate anti-proliferative effects of DAC treatment in 3D-models, indicated by a dose- and time-dependent reduction of EdU staining in CaSki and SiHa tumor spheroids and reduced p16INK4a and Ki-67 expression in the CaSki OTC model, which was accompanied by a significant reduction of tumor cell invasiveness.
Demethylating treatment represents a promising novel therapeutic strategy for HPV-transformed (pre-)cancerous lesions.