Bernard W Futscher

PMID: 17057734;Abstract:

The controlled and specific re-activation of endogenous tumor suppressors in cancer cells represents an important therapeutic strategy to block tumor growth and subsequent progression. Other than ectopic delivery of tumor suppressor-encoded cDNA, there are no therapeutic tools able to specifically re-activate tumor suppressor genes that are silenced in tumor cells. Herein, we describe a novel approach to specifically regulate dormant tumor suppressors in aggressive cancer cells. We have targeted the Mammary Serine Protease Inhibitor (maspin) (SERPINB5) tumor suppressor, which is silenced by transcriptional and aberrant promoter methylation in aggressive epithelial tumors. Maspin is a multifaceted protein, regulating tumor cell homeostasis through inhibition of cell growth, motility and invasion. We have constructed artificial transcription factors (ATFs) made of six zinc-finger (ZF) domains targeted against 18-base pair (bp) unique sequences in the maspin promoter. The ZFs were linked to the activator domain VP64 and delivered in breast tumor cells. We found that the designed ATFs specifically interact with their cognate targets in vitro with high affinity and selectivity. One ATF was able to re-activate maspin in cell lines that comprise a maspin promoter silenced by epigenetic mechanisms. Consistently, we found that this ATF was a powerful inducer of apoptosis and was able to knock down tumor cell invasion in vitro. Moreover, this ATF was able to suppress MDA-MB-231 growth in a xenograft breast cancer model in nude mice. Our work suggests that ATFs could be used in cancer therapeutics as novel molecular switches to re-activate dormant tumor suppressors. © 2007 Nature Publishing Group All rights reserved.

PMID: 18922938;PMCID: PMC2680223;Abstract:

Changes in DNA methylation patterns are a common characteristic of cancer cells. Recent studies suggest that DNA methylation affects not only discrete genes, but it can also affect large chromosomal regions, potentially leading to LRES. It is unclear whether such long-range epigenetic events are relatively rare or frequent occurrences in cancer. Here, we use a high-resolution promoter tiling array approach to analyze DNA methylation in breast cancer specimens and normal breast tissue to address this question. We identified 3,506 cancer-specific differentially methylated regions (DMR) in human breast cancer with 2,033 being hypermethylation events and 1,473 hypomethylation events. Most of these DMRs are recurrent in breast cancer; 90% of the identified DMRs occurred in at least 33% of the samples. Interestingly, we found a nonrandom spatial distribution of aberrantly methylated regions across the genome that showed a tendency to concentrate in relatively small genomic regions. Such agglomerates of hypermethylated and hypomethylated DMRs spanned up to several hundred kilobases and were frequently found at gene family clusters. The hypermethylation events usually occurred in the proximity of the transcription start site in CpG island promoters, whereas hypomethylation events were frequently found in regions of segmental duplication. One example of a newly discovered agglomerate of hypermethylated DMRs associated with gene silencing in breast cancer that we examined in greater detail involved the proto-cadherin gene family clusters on chromosome 5 (PCDHA, PCDHB, and PCDHG). Taken together, our results suggest that agglomerative epigenetic aberrations are frequent events in human breast cancer. ©2008 American Association for Cancer Research.

Abstract:

Genome-wide disruption of the epigenetic code is a hallmark of malignancy that encompasses many distinct, highly interactive modifications. Delineating the aberrant epigenome produced during toxicant-mediated malignant transformation will help identify the underlying epigenetic drivers of environmental toxicant-induced carcinogenesis. Gene promoter DNA methylation and gene expression profiling of arsenite-transformed prostate epithelial cells showed a negative correlation between gene expression changes and DNA methylation changes; however, less than 10% of the genes with increased promoter methylation were downregulated. Studies described herein confirm that a majority of the DNA hypermethylation events occur at H3K27me3 marked genes that were already transcriptionally repressed. In contrast to aberrant DNA methylation targeting H3K27me3 pre-marked silent genes, we found that actively expressed C2H2 zinc finger genes (ZNFs) marked with H3K9me3 on their 3' ends, were the favored targets of DNA methylation linked gene silencing. DNA methylation coupled, H3K9me3 mediated gene silencing of ZNF genes was widespread, occurring at individual ZNF genes on multiple chromosomes and across ZNF gene family clusters. At ZNF gene promoters, H3K9me3 and DNA hypermethylation replaced H3K4me3, resulting in a widespread downregulation of ZNF gene expression, which accounted for 8% of all the downregulated genes in the arsenical-transformed cells. In summary, these studies associate toxicant exposure with widespread silencing of ZNF genes by DNA hypermethylation-linked H3K9me3 spreading, further implicating epigenetic dysfunction as a driver of toxicant associated carcinogenesis. © 2013 Landes Bioscience.

PMID: 2311191;Abstract:

An attempt was made to characterize the genetic regulation of the human DNA repair protein O⁶-alkylguanine-DNA alkyltransferase (AGT) in the absence of the cloned gene. Four human cell lines, differing in AGT activity from very proficient to essentially absent, were assayed for gene amplification as a possible mediator of the methylation repair phenotype (Mer+, AGT activity and MER-, no AGT activity) using in-gel DNA renaturation and G-banded karyotype analysis. The former technique allows subsequent analysis of amplification units and cloning of observed amplified DNA fragments, a hopeful approach to the isolation of the human AGT gene. Within the sensitivities of the techniques, no correlation between AGT activity and gene amplification was observed in the four cell lines tested.