title: Functional validation of putative tumor suppressor gene C13ORF18 in cervical cancer by Artificial Transcription Factors.
abstract: C13ORF18 is frequently hypermethylated in cervical cancer but not in normal cervix and might serve as a biomarker for the early detection of cervical cancer in scrapings. As hypermethylation is often observed for silenced tumor suppressor genes (TSGs), hypermethylated biomarker genes might exhibit tumor suppressive activities upon re-expression. Epigenetic drugs are successfully exploited to reverse TSG silencing, but act genome-wide. Artificial Transcription Factors (ATFs) provide a gene-specific approach for re-expression of silenced genes. Here, we investigated the potential tumor suppressive role of C13ORF18 in cervical cancer by ATF-induced re-expression. Five zinc finger proteins were engineered to bind the C13ORF18 promoter and fused to a strong transcriptional activator. C13ORF18 expression could be induced in cervical cell lines: ranging from >40-fold in positive (C13ORF18-unmethylated) cells to >110-fold in negative (C13ORF18-methylated) cells. Re-activation of C13ORF18 resulted in significant cell growth inhibition and/or induction of apoptosis. Co-treatment of cell lines with ATFs and epigenetic drugs further enhanced the ATF-induced effects. Interestingly, re-activation of C13ORF18 led to partial demethylation of the C13ORF18 promoter and decreased repressive histone methylation. These data demonstrate the potency of ATFs to re-express and potentially demethylate hypermethylated silenced genes. Concluding, we show that C13ORF18 has a TSG function in cervical cancer and may serve as a therapeutic anti-cancer target. As the amount of epimutations in cancer exceeds the number of gene mutations, ATFs provide promising tools to validate hypermethylated marker genes as therapeutic targets.
authors.affiliation: Dept. of Pathology and Medical Biology, University Medical Centre Groningen (UMCG), University of Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. c.huisman@umcg.nl
authors.forename: Christian
authors.initials: C
authors.lastname: Huisman
authors.forename: G Bea A
authors.initials: GB
authors.lastname: Wisman
authors.forename: Hinke G
authors.initials: HG
authors.lastname: Kazemier
authors.forename: Marcel A T M
authors.initials: MA
authors.lastname: van Vugt
authors.forename: Ate G J
authors.initials: AG
authors.lastname: van der Zee
authors.forename: Ed
authors.initials: E
authors.lastname: Schuuring
authors.forename: Marianne G
authors.initials: MG
authors.lastname: Rots
mesh_terms: D045744:Cell Line, Tumor; D002584:Cervix Uteri / Q000378:metabolism / Q000473:pathology*; D019175:DNA Methylation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D016147:Genes, Tumor Suppressor; D006801:Humans; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000473:pathology
chemical_list: C585115:RUBCNL protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins