title: Re-expression of Selected Epigenetically Silenced Candidate Tumor Suppressor Genes in Cervical Cancer by TET2-directed Demethylation.
abstract: DNA hypermethylation is extensively explored as therapeutic target for gene expression modulation in cancer. Here, we re-activated hypermethylated candidate tumor suppressor genes (TSGs) (C13ORF18, CCNA1, TFPI2, and Maspin) by TET2-induced demethylation in cervical cancer cell lines. To redirect TET2 to hypermethylated TSGs, we engineered zinc finger proteins (ZFPs), which were first fused to the transcriptional activator VP64 to validate effective gene re-expression and confirm TSG function. ChIP-Seq not only revealed enriched binding of ZFPs to their intended sequence, but also considerable off-target binding, especially at promoter regions. Nevertheless, results obtained by targeted re-expression using ZFP-VP64 constructs were in line with cDNA overexpression; both revealed strong growth inhibition for C13ORF18 and TFPI2, but not for CCNA1 and Maspin. To explore effectivity of locus-targeted demethylation, ZFP-TET2 fusions were constructed which efficiently demethylated genes with subsequent gene re-activation. Moreover, targeting TET2 to TFPI2 and C13ORF18, but not CCNA1, significantly decreased cell growth, viability, and colony formation in cervical cancer cells compared to a catalytically inactive mutant of TET2. These data underline that effective re-activation of hypermethylated genes can be achieved through targeted DNA demethylation by TET2, which can assist in realizing sustained re-expression of genes of interest.
authors.affiliation: Department of Pathology and Medical Biology, University Medical Center Groningen (UMCG), University of Groningen, Groningen, The Netherlands.|Current address: Department of Pediatrics, Oregon Health and Science University, Portland, Oregon, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Huisman
authors.affiliation: Department of Pathology and Medical Biology, University Medical Center Groningen (UMCG), University of Groningen, Groningen, The Netherlands.
authors.forename: Monique G P
authors.initials: MG
authors.lastname: van der Wijst
authors.affiliation: Department of Pathology and Medical Biology, University Medical Center Groningen (UMCG), University of Groningen, Groningen, The Netherlands.
authors.forename: Matthijs
authors.initials: M
authors.lastname: Schokker
authors.affiliation: Cancer Epigenetics Group, The Harry Perkins Institute for Medical Research, The University of Western Australia, Nedlands, Western Australia, Australia.
authors.forename: Pilar
authors.initials: P
authors.lastname: Blancafort
authors.affiliation: Department of Genetics, University Medical Center Groningen (UMCG), University of Groningen, Groningen, The Netherlands.
authors.forename: Martijn M
authors.initials: MM
authors.lastname: Terpstra
authors.affiliation: Department of Genetics, University Medical Center Groningen (UMCG), University of Groningen, Groningen, The Netherlands.
authors.forename: Klaas
authors.initials: K
authors.lastname: Kok
authors.affiliation: Department of Gynecological Oncology, University Medical Center Groningen (UMCG), University of Groningen, Groningen, The Netherlands.
authors.forename: Ate G J
authors.initials: AG
authors.lastname: van der Zee
authors.affiliation: Department of Pathology and Medical Biology, University Medical Center Groningen (UMCG), University of Groningen, Groningen, The Netherlands.
authors.forename: Ed
authors.initials: E
authors.lastname: Schuuring
authors.affiliation: Department of Gynecological Oncology, University Medical Center Groningen (UMCG), University of Groningen, Groningen, The Netherlands.
authors.forename: G Bea A
authors.initials: GB
authors.lastname: Wisman
authors.affiliation: Department of Pathology and Medical Biology, University Medical Center Groningen (UMCG), University of Groningen, Groningen, The Netherlands.
authors.forename: Marianne G
authors.initials: MG
authors.lastname: Rots
mesh_terms: D045744:Cell Line, Tumor; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D049308:Dioxygenases; D044127:Epigenesis, Genetic; D005260:Female; D000072669:Gene Editing; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D016147:Genes, Tumor Suppressor; D006801:Humans; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000378:metabolism*; D016335:Zinc Fingers
chemical_list: D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D049308:Dioxygenases; C541081:TET2 protein, human