High Expression of hTERT and Stemness Genes in BORIS/CTCFL Positive Cells Isolated from Embryonic Cancer Cells

BORIS/CTCFL is a member of cancer testis antigen family normally expressed in germ cells. In tumors, it is aberrantly expressed although its functions are not completely well-defined. To better understand the functions of BORIS in cancer, we selected the embryonic cancer cells as a model. Using a molecular beacon, which specifically targets BORIS mRNA, we demonstrated that BORIS positive cells are a small subpopulation of tumor cells (3–5% of total). The BORIS-positive cells isolated using BORIS-molecular beacon, expressed higher telomerase hTERT, stem cell (NANOG, OCT4, SOX2) and cancer stem cell marker genes (CD44 and ALDH1) compared to the BORIS-negative tumor cells. In order to define the functional role of BORIS, stable BORIS-depleted embryonic cancer cells were generated. BORIS silencing strongly down-regulated the expression of hTERT, stem cell and cancer stem cell marker genes. Moreover, the BORIS knockdown increased cellular senescence in embryonic cancer cells, revealing a putative role of BORIS in the senescence biological program. Our data indicate an association of BORIS expressing cells subpopulation with the expression of stemness genes, highlighting the critical role played by BORIS in embryonic neoplastic disease.     

BAT3 and SET1A form a complex with CTCFL/BORIS to modulate H3K4 histone dimethylation and gene expression

Chromatin status is characterized in part by covalent posttranslational modifications of histones that regulate chromatin dynamics and direct gene expression. BORIS (brother of the regulator of imprinted sites) is an insulator DNA-binding protein that is thought to play a role in chromatin organization and gene expression. BORIS is a cancer-germ line gene; these are genes normally present in male germ cells (testis) that are also expressed in cancer cell lines as well as primary tumors. This work identifies SET1A, an H3K4 methyltransferase, and BAT3, a cochaperone recruiter, as binding partners for BORIS, and these proteins bind to the upstream promoter regions of two well-characterized procarcinogenic genes, Myc and BRCA1. RNA interference (RNAi) knockdown of BAT3, as well as SET1A, decreased Myc and BRCA1 gene expression but did not affect the binding properties of BORIS, but RNAi knockdown of BORIS prevented the assembly of BAT3 and SET1A at the Myc and BRCA1 promoters. Finally, chromatin analysis suggested that BORIS and BAT3 exert their effects on gene expression by recruiting proteins such as SET1A that are linked to changes in H3K4 dimethylation. Thus, we propose that BORIS acts as a platform upon which BAT3 and SET1A assemble and exert effects upon chromatin structure and gene expression.     

Aberrant alternative splicing of RHOA is associated with loss of its expression and activity in diffuse-type gastric carcinoma cells

RhoA is a member of Rho family small GTPases that regulates diverse cellular functions. Recent large-scale sequencing studies have identified recurrent somatic mutations of RHOA in diffuse-type gastric carcinoma (DGC), indicating that RHOA is a driver of DGC. In this study, we investigated the possible abnormalities of RHOA in a panel of gastric carcinoma (GC) cell lines. Pulldown assay and immunoblot analysis showed that the activity and expression of RhoA were detectable in all GC cell lines tested, except for two DGC cell lines, HSC-59 and GSU. RHOA coding region sequencing revealed that aberrant alternative splicing of RHOA occurred in these cell lines. Quantitative real-time PCR analysis showed that the expression of wild-type RHOA was nearly undetectable, whereas splicing variants were almost exclusively expressed in HSC-59 and GSU cell lines. However, the expression levels of RHOA splicing variants were very low and the corresponding proteins were not detected by immunoblotting. Moreover, the splicing isoforms of RhoA protein were neither efficiently expressed nor activated even if ectopically expressed in cells. These results indicate that aberrant alternative splicing of RHOA results in the loss of its activity and expression in DGC cells.