Abstract 3146: Awake the sleeping: Gene specific re-expression of epigenetically silenced candidate tumor suppressor genes in cervical cancer by artificial transcription factors
Authors: Huisman Christian , Wisman Bea A. , Falahi Fahimeh , Kazemier Hinke G. , Schuuring Ed , van der Zee Ate GJ , Rots Marianne G. 
Published: August 2012

Abstract
Recently, we reported diagnostic marker gene promoters that are hypermethylated in cervical cancer. As hypermethylation is often seen for tumor suppressor genes (TSGs), such marker genes might exhibit tumor suppressive activities upon re-expression. In this study, we aim to specifically upregulate candidate TSGs (C13ORF18, EPB41L3 and CCNA1) using self-designed Artificial Transcription Factors (ATFs). An advantage of this approach is that genes are re-expressed from their natural promoter allowing all splice variants to be expressed in natural ratios In cervical cancer cell lines, DNA methylation status (methylation-specific PCR, bisulfite sequencing) and gene (re)expression levels (qRT-PCR) were determined, also after treatment with a DNA demethylating agent (5-Aza-2′-Deoxycytidine (DAC)) and a histone deacetylase inhibitor (Trichostatin A (TSA)). Sequence specific DNA-binding domains were constructed by engineering zinc finger proteins and fused to a strong transcriptional activator. Functional effects of re-expression of the genes were studied using cell survival/apoptosis/cell growth assays. Histone marks were identified using chromatin IP, before and after treatment with ATFs. C13ORF18, EPB41L3 and CCNA1 silencing in cervical cancer was associated with DNA hypermethylation and these genes could be re-expressed in a dose dependent manner using DAC. Gene-specific re-activation of the genes could be achieved by the ATFs: Up to 300-fold in negative (methylated) cells and up to 80-fold in positive (unmethylated) cells. Co-treatment of ATFs with DAC or TSA further enhanced the effect of the ATFs. The hypermethylation status of the genes was associated with low levels of histone acetylation and repressive histone marks (H3K9me3, H3K27me3). Interestingly, preliminary data show that re-activation of the silenced genes increased the histone acetylation, while repressive histone marks decreased, indicating that ATF-induced re-expression was associated with changes in the epigenetic code. Furthermore, it was found that specific re-activation of the genes inhibited cell growth and induced apoptosis. These data confirm our hypothesis that hypermethylated cancer-specific genes can be re-activated, resulting in a strong anti-tumor effect. Furthermore, this is the first time that these genes are reported as TSGs in cervical cancer. The great advantage of gene-specific DNA targeting is that only the TSGs of interest are upregulated, while current genome-wide epigenetic drugs also target metastasis inducing genes, and can therefore enforce cancer growth. Currently, the DNA binding domains are fused to epigenetic effector domains to explore the ability of gene specific epigenetic editing. Financed by NWO-VIDI to MR.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3146. doi:1538-7445.AM2012-3146