title: A recombinant reporter system for monitoring reactivation of an endogenously DNA hypermethylated gene.
abstract: Reversing abnormal gene silencing in cancer cells due to DNA hypermethylation of promoter CpG islands may offer new cancer prevention or therapeutic approaches. Moreover, such approaches may be broadly applicable to enhance the efficacy of radiotherapy, chemotherapy, or immunotherapy. Here, we demonstrate the powerful utility of a novel gene reporter system to permit studies of the dynamics, mechanisms, and translational relevance of candidate therapies of this type in human colon cancer cells. The reporter system is based on in situ modification of the endogenous locus of the tumor-suppressor gene SFRP1, a pivotal regulator of the Wnt pathway that is silenced by DNA hypermethylation in many colon cancers. The modified SFRP1-GFP reporter allele used remained basally silent, like the unaltered allele, and it was activated only by drug treatments that derepress gene silencing by reversing DNA hypermethylation. We used the established DNA methyltransferase inhibitor (DNMTi) 5-aza-deoxycitidine (DAC) to show how this system can be used to address key questions in the clinical development of epigenetic cancer therapies. First, we defined conditions for which clinically relevant dosing could induce sustained induction of RNA and protein. Second, we found that, in vivo, a more prolonged drug exposure than anticipated was essential to derepress gene silencing in significant cell numbers, and this has implications for generating effective anticancer responses in patients with hematopoietic or solid tumors. Finally, we discovered how histone deacetylase inhibitors (HDACi) alone, when administered to cells actively replicating DNA, can robustly reexpress the silenced gene with no change in promoter methylation status. Taken together, our findings offer a new tool and insights for devising optimal clinical experiments to evaluate DNMTi and HDACi, alone or in combination, and with other cancer treatments, as agents for the epigenetic management and prevention of cancer.
authors.affiliation: Authors' Affiliations: Cancer Biology Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins;
authors.forename: Ying
authors.initials: Y
authors.lastname: Cui
authors.affiliation: BioNumerik Pharmaceuticals, Inc., San Antonio, Texas; and.
authors.forename: Frederick
authors.initials: F
authors.lastname: Hausheer
authors.affiliation: Authors' Affiliations: Cancer Biology Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins;
authors.forename: Robert
authors.initials: R
authors.lastname: Beaty
authors.affiliation: Authors' Affiliations: Cancer Biology Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins;
authors.forename: Cynthia
authors.initials: C
authors.lastname: Zahnow
authors.affiliation: Fels Institute for Cancer and Molecular Biology, Temple University, Philadelphia, Pennsylvania.
authors.forename: Jean Pierre
authors.initials: JP
authors.lastname: Issa
authors.affiliation: Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland;
authors.forename: Frederick
authors.initials: F
authors.lastname: Bunz
authors.affiliation: Authors' Affiliations: Cancer Biology Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins; sbaylin@jhmi.edu.
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Baylin
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation; D004195:Disease Models, Animal; D005260:Female; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D023061:Gene Order; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D064593:Heterografts; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics; D015533:Transcriptional Activation; D000094742:Secreted Frizzled-Related Proteins
chemical_list: D001374:Azacitidine; D049452:Green Fluorescent Proteins; D056572:Histone Deacetylase Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C117636:SFRP1 protein, human; D000094742:Secreted Frizzled-Related Proteins