Introduction:
Decitabine (DAC) treatment induces a genome wide reduction of DNA methylation. Cancer-testis antigen (CTA) expression is regulated via gene promoter methylation. Demethylating agents have been shown to increase CTA expression, which has been implied to improve CTA-specific immunotherapy. Our main objective was to investigate the effect of DAC on CTA expression in head and neck cancer cell lines (HNSCC) in vitro, as well as the correlation between promoter methylation and RNA levels of CTA.
Methods:
Head and neck cancer cell lines (four HPV+ and four HPV- cell lines) were treated with 2µM DAC for 5 consecutive days. Cells were harvested thereafter, DNA and RNA were isolated and prepared for pyrosequencing and real-time PCR of CTA genes (MAGE-A3, NY-ESO-1 and PRAME) in comparison to untreated cells. Cell proliferation was measured using the xCELLigence RTCA DP instrument.
Results:
DAC treatment resulted in increased expression of MAGE-A3, NY-ESO-1 and PRAME in all cell lines. Whereas for MAGE-A3 and NY-ESO-1 increased expression was associated with decreased gene promoter methylation, this was not the case for PRAME. Untreated HPV+ cell lines expressed very low levels of all three CTA genes compared to HPV- cell lines, but DAC treatment significantly increased CTA expression. DAC did not affect proliferation significantly.
Conclusions:
Expression and methylation of CTA genes differ between HPV+ and HPV- cell lines. We showed DNA gene promoter demethylation of MAGE-A3 and NY-ESO-1 is associated with overexpression on RNA level. Since DAC is an unspecific demethylator, demethylation of Enhancers may contribute to overexpression. Upregulation of CTA expression by DAC treatment may increase immunogenicity of cancer cells.
