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)

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)

An immunogenic murine fibrosarcoma cell line was genetically modified to express and produce the human RANTES chemokine stably. In in vitro chemotaxis assays purified recombinant human RANTES as well as human RANTES secreted by the modified murine tumor cells were strongly chemoattractant for mouse CD8+ /Thy-1+ tumor-infiltrating lymphocytes (TIL). RANTES production did not alter the growth of these cytokine gene-modified tumor cells in vitro, but injection of RANTES-secreting cells resulted in the abolition of the ability of those cells to form solid tumors in vivo. The growth of tumors could be restored by co-administration of monoclonal antibodies that inhibit the function of various subsets of immune cells. For example, depletion of CD8+ T cells by antibody administration resulted in complete restoration of solid tumor formation by RANTES-secreting cells, whereas depletion of the CD4+ T cell population resulted in a partial restoration of tumor formation. Additionally, administration of an anti-CR3 monoclonal antibody known to inhibit the in vivo migration of macrophages also completely restored the tumorigenicity of RANTES-secreting fibrosarcoma cells. Thus, the human RANTES chemokine can abolish tumorigenicity of an immunogenic fibrosarcoma in an in vivo murine model, and this process is mediated by various subpopulations of immune effector cells.

Purpose: Cancer/testis (CT) genes predominantly expressed in the testis (germ cells) and generally not in other normal tissues are aberrantly expressed in human cancers. This highly restricted expression provides a unique opportunity to use these CTgenes for diagnostics, immunotherapeutic, or other targeted therapies. The purpose of this study was to identify those CT genes with the greatest incidence of expression in uterine cancers. Experimental Design: We queried the expression of known and putative CT gene transcripts (representing 79 gene loci) using whole genome gene expression arrays. Specifically, the global gene expressions of uterine cancers (n = 122) and normal uteri (n = 10) were determined using expression data from the Affymetrix HG-U133A and HG-U133B chips. Additionally, we also examined the brother of the regulator of imprinted sites (BORIS) transcript by reverse transcription-PCR and quantitative PCR because its transcript was not represented on the array. Results: Global microarray analysis detected many CT genes expressed in various uterine cancers; however, no individual CT gene was expressed in more than 25% of all cancers. The expression of the two most commonly expressed CT genes on the arrays, MAGEA9 (24 of 122 cancers and 0 of10 normal tissues) and Down syndrome critical region 8 (DSCR8)/MMA1 (16 if 122 cancers and 0 of 10 normal tissues), was confirmed by reverse transcription-PCR methods, validating the array screening approach. In contrast to the relatively low incidence of expression of the other CTgenes, BORIS expression was detected in 73 of 95 (77%) endometrial cancers and 24 of 31 (77%) uterine mixed mesodermal tumors. Conclusions: These data provide the first extensive survey of multiple CT genes in uterine cancers. Importantly, we detected a high frequency of BORIS expression in uterine cancers, suggesting its potential as an immunologic or diagnostic target for these cancers. Given the high incidence of BORIS expression and its possible regulatory role, an examination of BORIS function in the etiology of these cancers is warranted.