Brother of the Regulator of Imprinted Sites (BORIS) is a mammalian CTCF paralog with the same central 11Zn fingers (11ZF) that mediate specific interactions with varying f50-bp target sites. Regulated in vivo occupancy of such sites may yield structurally and functionally distinct CTCF/DNA complexes involved in various aspects of gene regulation, including epigenetic control of gene imprinting and X chromosome inactivation. The latter functions are mediated by meCpGsensitive 11ZF binding. Because CTCF is normally present in all somatic cells, whereas BORIS is active only in CTCF-and 5-methylcytosine-deficient adult male germ cells, switching DNA occupancy from CTCF to BORIS was suggested to regulate site specificity and timing of epigenetic reprogramming. In addition to 11ZF-binding paternal imprinting control regions, cancer-testis gene promoters also undergo remethylation during CTCF/BORIS switching in germ cells. Only promoters of cancer testis genes are normally silenced in all somatic cells but activated during spermatogenesis when demethylated in BORIS-positive germ cells and are found aberrantly derepressed in various tumors. We show here that BORIS is also expressed in multiple cancers and is thus itself a cancer-testis gene and that conditional expression of BORIS in normal fibroblasts activates cancer-testis genes selectively. We tested if replacement of CTCF by BORIS on regulatory DNA occurs in vivo on activation of a prototype cancer-testis gene, MAGE-A1. Transition from a hypermethylated/silenced to a hypomethylated/activated status induced in normal cells by 5-aza-2V -deoxycytidine (5-azadC) was mimicked by conditional input of BORIS and is associated with complete switching from CTCF to BORIS occupancy at a single 11ZF target. This site manifested a novel type of CTCF/BORIS 11ZF binding insensitive to CpG methylation. Whereas 5-azadC induction of BORIS takes only few hours, derepression of MAGE-A1 occurred 1 to 2 days later, suggesting that BORIS mediates cancer-testis gene activation by 5-azadC. Indeed, infection of normal fibroblasts with anti-BORIS short hairpin RNA retroviruses before treatment with 5-azadC blocked reactivation of MAGE-A1. We suggest that BORIS is likely tethering epigenetic machinery to a novel class of CTCF/BORIS 11ZF target sequences that mediate induction of cancer-testis genes. (Cancer Res 2005; 65(17): 7751-62)

Limited information is available regarding epigenomic events mediating initiation and progression of tobacco-induced lung cancers. In this study, we established an in vitro system to examine epigenomic effects of cigarette smoke in respiratory epithelia. Normal human small airway epithelial cells and cdk-4/hTERT-immortalized human bronchial epithelial cells (HBEC) were cultured in normal media with or without cigarette smoke condensate (CSC) for up to 9 months under potentially relevant exposure conditions. Western blot analysis showed that CSC mediated dose-and timedependent diminution of H4K16Ac and H4K20Me3, while increasing relative levels of H3K27Me3; these histone alterations coincided with decreased DNA methyltransferase 1 (DNMT1) and increased DNMT3b expression. Pyrosequencing and quantitative RT-PCR experiments revealed time-dependent hypomethylation of D4Z4, NBL2, and LINE-1 repetitive DNA sequences; up-regulation of H19, IGF2, MAGE-A1, and MAGE-A3; activation of Wnt signaling; and hypermethylation of tumor suppressor genes such as RASSF1A and RAR-b, which are frequently silenced in human lung cancers. Array-based DNA methylation profiling identified additional novel DNA methylation targets in soft-agar clones derived from CSC-exposed HBEC; a CSC gene expression signature was also identified in these cells. Progressive genomic hypomethylation and locoregional DNA hypermethylation induced by CSC coincided with a dramatic increase in soft-agar clonogenicity. Collectively, these data indicate that cigarette smoke induces 'cancer-associated' epigenomic alterations in cultured respiratory epithelia. This in vitro model may prove useful for delineating early epigenetic mechanisms regulating gene expression during pulmonary carcinogenesis.

Regulatory sequences recognized by the unique pair of paralogous factors, CTCF and BORIS, have been implicated in epigenetic regulation of imprinting and X chromosome inactivation. Lung cancers exhibit genome-wide demethylation associated with derepression of a specific class of genes encoding cancer-testis (CT) antigens such as NY-ESO-1. CT genes are normally expressed in BORIS-positive male germ cells deficient in CTCF and meCpG contents, but are strictly silenced in somatic cells. The present study was undertaken to ascertain if aberrant activation of BORIS contributes to derepression of NY-ESO-1 during pulmonary carcinogenesis. Preliminary experiments indicated that NY-ESO-1 expression coincided with derepression of BORIS in cultured lung cancer cells. Quantitative reverse transcription-PCR analysis revealed robust, coincident induction of BORIS and NY-ESO-1 expression in lung cancer cells, but not normal human bronchial epithelial cells following 5-aza-2V -deoxycytidine (5-azadC), Depsipeptide FK228 (DP), or sequential 5-azadC/DP exposure under clinically relevant conditions. Bisulfite sequencing, methylation-specific PCR, and chromatin immunoprecipitation (ChIP) experiments showed that induction of BORIS coincided with direct modulation of chromatin structure within a CpG island in the 5V -flanking noncoding region of this gene. Cotransfection experiments using promoter-reporter constructs confirmed that BORIS modulates NY-ESO-1 expression in lung cancer cells. Gel shift and ChIP experiments revealed a novel CTCF/BORIS-binding site in the NY-ESO-1 promoter, which unlike such sites in the H19-imprinting control region and X chromosome, is insensitive to CpG methylation in vitro. In vivo occupancy of this site by CTCF was associated with silencing of the NY-ESO-1 promoter, whereas switching from CTCF to BORIS occupancy coincided with derepression of NY-ESO-1. Collectively, these data indicate that reciprocal binding of CTCF and BORIS to the NY-ESO-1 promoter mediates epigenetic regulation of this CT gene in lung cancer cells, and suggest that induction of BORIS may be a novel strategy to augment immunogenicity of pulmonary carcinomas. (Cancer Res 2005; 65(17): 7763-74)

Purpose: The DNA methylation paradox, manifested as derepression of cancer-testis antigens, and silencing of tumor suppressors during malignant transformation, provides the rationale for the utilization of chromatin remodeling agents for cancer therapy. A phase I trial was done to examine pharmacokinetics, toxicities, and gene expression mediated by 5-aza-2 ¶-deoxycytidine (DAC) in patients with thoracic malignancies. Experimental Design: Thirty-five patients with cancers refractory to standard therapy received continuous 72-hour DAC infusions using a phase I dose-escalation schema. Each full course of therapy consisted of two identical 35-day cycles. Plasma DAC levels were evaluated by liquid chromatography-mass spectrometry techniques. Quantitative reverse transcription-PCR, methylation-specific PCR, and immunohistochemical techniques were used to evaluate NY-ESO-1, MAGE-3, and p16 expression in tumor biopsies. Long oligonucleotide arrays were used to evaluate gene expression profiles in laser-captured tumor cells before and after DAC exposure. Results: Thirty-five patients were evaluable for toxicities; 25 were evaluable for treatment response. Myelosuppression constituted dose-limiting toxicity. The maximum tolerated dose of DAC was 60 to 75 mg/m 2 depending on the number of prior cytotoxic chemotherapy regimens. No objective responses were observed. Plasma DAC concentrations approximated thresholds for gene induction in cultured cancer cells. Target gene induction was observed in 36% of patients. Posttreatment antibodies to NY-ESO-1 were detected in three patients exhibiting NY-ESO-1 induction in their tumor tissues. Complex, heterogeneous gene expression profiles were observed in pretreatment and posttreatment tissues. Conclusion: Prolonged DAC infusions can modulate gene expression in primary thoracic malignancies. These findings support further evaluation of DNA-demethylating agents alone or in combination with other regimens targeting induced gene products for the treatment of these neoplasms.