(1) BORIS and cancer. During the last year, we advanced our studies of BORIS (Brother Of the Regulator of Imprinted Sites) - a CTCF-paralog, which we discovered. BORIS shares with CTCF a nearly identical 11 Zn-finger (11ZF) DNA binding domain (DBD), but their flanking NH2- and COOH-terminal regions are divergent. The 11ZF region was previously identified in the lab as a multivalent DBD, which is able to recognize and bind extended (around 50bp) target sequences. By virue of sharing the identical DBD, CTCF and BORIS can recignize the same DNA sequences, but likely have distinct regulation and form different associations with protein cofactors. Furthermore, due to the tissue-specific expression of BORIS in male germ cells, it is likely involved in re-establishment of paternal-specific DNA methylation patterns at particular imprinted sites of the Igf2/H19 locus through specific loop formation, by utilizing novel CTCF/BORIS sites. Based on our studies we predicted that most ICR sequences would contain meCpG-sensitive CTCF/BORIS target sites, which was validated for several unrelated imprinted loci. In addition to its role in development, BORIS likely plays a key role in oncogenesis. Indeed, while BORIS expression is silenced in normal somatic cells, it is activated in cancer cells;i.e. BORIS is a so-called cancer-testis (CT) gene. We and others previously characterized BORIS expression in uterine cancers, breast cancers, osteosarcomas, lung cancers, and prostate cancers. However, as BORIS is itself a gene expression regulator, it was hypothesized that BORIS-mediated control of promoters is the regulatory network responsible for the expression of multiple CT genes. Recently, we conducted a comprehensive functional analysis of BORIS expression in primary head and neck squamous cell carcinoma (HNSCC) as well as pharmacologically demethylated cell lines to identify aberrantly demethylated and expressed candidate proto-oncogenes and cancer testes antigens in HNSCC. The results of this study implicate BORIS in the reactivation of epigenetically silenced genes in human cancers via coordinated promoter demethylation. Aberrant BORIS expression specifically correlated with upregulation of candidate proto-oncogenes in multiple human malignancies including primary non-small cell lung cancers and HNSCC. Furthermore, BORIS activation induced coordinated proto-oncogene-specific promoter demethylation and expression in non-tumorigenic cells, as well as transformed NIH3T3 cells. These results elucidate the mechanism of relaxation of silencing of BORIS-regulated promoters following loss of functional p53. As the first step, we employed a comparative epigenetic approach utilizing Cancer Outlier Profiling Analysis (COPA) to test 49 primary HNSCC and 19 normal mucosal tissues for mRNA expression levels (the Affymetrix U133A mRNA expression microarray, 16,383 probe sets). Using statistical analysis we identified 106 genes that were significantly upregulated in cancer cells. The top scoring 26 genes were selected for further analyses, and 17 of them were discovered to contain promoter-associated CpG islands. In a parallel experiment we analyzed 32,500 genes (U133plus2 microarray) with respect to their upregulation in response to 5-aza/TSA treatment of normalized cell lines that were not included in primary tumor expression analysis. Among 46 target genes identified, 30 were confirmed to have CpG islands. Transcriptional upregulation of a 9 target gene sample after 5-aza/TSA treatment was then confirmed by quantitative RT-PCR and functionally - by transient transfections of the corresponding genes. Among such genes were MAGEA2, MAGEA3/6, MAGEA11, TKTL1, MAGEA4, C19ORF28, and GRIN1. As the presence of several MAGE genes targets indicated that BORIS or CTCF may be involved in such an epigenetic regulation, we assessed BORIS expression in a separate sample of 36 primary HNSCC. BORIS overexpression was significantly correlated with overexpression of 6 proto-oncogenes including: MAGEA3/6, MAGEA4, MAGEA11, GPR17, and C19ORF28. Furthermore, analysis of public cancer databases revealed that 59% of all tumors have BORIS levels exceeding the median expression of all genes, and 90% of tumors have BORIS expression level exceedind at least . of median expression for all genes. These data confirm that aberrant BORIS expression is a universal feature of human cancers. Finally, we used tetracycline-inducible pBIG2i-BORIS constructs transiently transfected into NIH-3T3 and OKF6-Tert1R cell lines to model the effect of BORIS on candidate target genes. Expression of seven of nine target genes was significantly increased in OKF6-Tert1R cell expressing BORIS, and six out of nine targets showed a greater than 100% increase in demethylated promoters 48 hours after induction of BORIS. Thus, BORIS is the key transcriptional regulator involved in the coordinated derepression of a of growth-promoting proto-oncogene candidates. Therefore, BORIS may present a promising therapeutic target for a directed effect on multiple oncogenic pathways. (2) Studies on BORIS role in non-cancer cells have also led to several significant findings. As a result, an Employee Invention Report (E-165-2009/0-EIR-00) was filed listing Svetlana Pack, Ziyedulla N. Abdullayev, and Victor V. Lobanenkov as inventors. NIAID has approved filing a patent application, which is planned for October, 2009.
Showing the most recent 10 out of 21 publications
